Absence Of Ataxia Telangiectasia Mutated Research Articles

Absence of ATM leads to altered NK cell function in mice

Ataxia-telangiectasia (A-T) is a rare disorder caused by genetic defects of A-T mutated (ATM) kinase, a key regulator of stress response, and characterized by neurodegeneration, immunodeficiency, and high incidence of cancer. Here we investigated NK cells in a mouse model of A-T (Atm−/−) showing that they are strongly impaired at killing tumor cells due to a block of early signaling events. On the other hand, in Atm−/− littermates with thymic lymphoma NK cell cytotoxicity is enhanced as compared with ATM-proficient mice, possibly via tumor-produced TNF-α. Results also suggest that expansion of exhausted NKG2D+ NK cells in Atm−/− mice is driven by low-level expression of stress-inducible NKG2D ligands, whereas development of thymoma expressing the high-affinity MULT1 ligand is associated with NKG2D down-regulation on NK cells.These results expand our understanding of immunodeficiency in A-T and encourage exploring NK cell biology in A-T patients in the attempt to identify cancer predictive biomarkers and novel therapeutic targets.

ATM supports gammaherpesvirus replication by attenuating type I interferon pathway

Ataxia-Telangiectasia mutated (ATM) kinase participates in multiple networks, including DNA damage response, oxidative stress, and mitophagy. ATM also supports replication of diverse DNA and RNA viruses. Gammaherpesviruses are prevalent cancer-associated viruses that benefit from ATM expression during replication. This proviral role of ATM had been ascribed to its signaling within the DNA damage response network; other functions of ATM have not been considered. In this study increased type I interferon (IFN) responses were observed in ATM deficient gammaherpesvirus-infected macrophages. Using a mouse model that combines ATM and type I IFN receptor deficiencies we show that increased type I IFN response in the absence of ATM fully accounts for the proviral role of ATM during gammaherpesvirus replication. Further, increased type I IFN response rendered ATM deficient macrophages more susceptible to antiviral effects of type II IFN. This study identifies attenuation of type I IFN responses as the primary mechanism underlying proviral function of ATM during gammaherpesvirus infection.

Infection of a Single Cell Line with Distinct Strains of Human Cytomegalovirus Can Result in Large Variations in Virion Production and Facilitate Efficient Screening of Virus Protein Function.

Previously, we reported that the absence of the ataxia telangiectasia mutated (ATM) kinase, a critical DNA damage response (DDR) signaling component for double-strand breaks, caused no change in HCMV Towne virion production. Later, others reported decreased AD169 viral titers in the absence of ATM. To address this discrepancy, human foreskin fibroblasts (HFF) and three ATM(-) lines (GM02530, GM05823, and GM03395) were infected with both Towne and AD169. Two additional ATM(-) lines (GM02052 and GM03487) were infected with Towne. Remarkably, both previous studies' results were confirmed. However, the increased number of cell lines and infections with both lab-adapted strains confirmed that ATM was not necessary to produce wild-type-level titers in fibroblasts. Instead, interactions between individual virus strains and the cellular microenvironment of the individual ATM(-) line determined efficiency of virion production. Surprisingly, these two commonly used lab-adapted strains produced drastically different titers in one ATM(-) cell line, GM05823. The differences in titer suggested a rapid method for identifying genes involved in differential virion production. In silico comparison of the Towne and AD169 genomes determined a list of 28 probable candidates responsible for the difference. Using serial iterations of an experiment involving virion entry and input genome nuclear trafficking with a panel of related strains, we reduced this list to four (UL129, UL145, UL147, and UL148). As a proof of principle, reintroduction of UL148 largely rescued genome trafficking. Therefore, use of a battery of related strains offers an efficient method to narrow lists of candidate genes affecting various virus life cycle checkpoints. Human cytomegalovirus (HCMV) infection of multiple cell lines lacking ataxia telangiectasia mutated (ATM) protein produced wild-type levels of infectious virus. Interactions between virus strains and the microenvironment of individual ATM(-) lines determined the efficiency of virion production. Infection of one ATM(-) cell line, GM05823, produced large titer differentials dependent on the strain used, Towne or AD169. This discrepancy resolved a disagreement in the literature of a requirement for ATM expression and HCMV reproduction. The titer differentials in GM08523 cells were due, in part, to a decreased capacity of AD169 virions to enter the cell and traffic genomes to the nucleus. In silico comparison of the Towne, AD169, and related variant strains' genomes was coupled with serial iterations of a virus entry experiment, narrowing 28 candidate proteins responsible for the phenotype down to 4. Reintroduction of UL148 significantly rescued genome trafficking. Differential behavior of virus strains can be exploited to elucidate gene function.

Individual Cytokines Modulate the Neurological Symptoms of ATM Deficiency in a Region Specific Manner

Ataxia-telangiectasia (A-T) is a multisystemic neurodegenerative disease of childhood caused by the absence of functional ATM (A-T mutated) protein. The cerebellar cortex has the most obvious neuropathology, yet cells in other brain regions are also abnormal. A-T mouse models have been produced that replicate much, though not all, of the complex A-T phenotype. Nongenetic factors, including modulations of the immune status of the animal, have also recently been found to play a role in the disease phenotype. Here we report that these modulations show both cytokine and brain region specificity. The CNS changes induced by broad-spectrum immune challenges, such as lipopolysaccharide (LPS) injections are a complex mixture of neuroprotective (TNFα) and neurodegenerative (IL1β) cytokine responses that change over time. For example, LPS first induces a protective response in A-T neurons through activation of tissue repair genes through infiltration of monocytes with M2 phenotype, followed over time by a set of more degenerative responses. Additional phenotypic complexity arises because the neuronal response to an immune challenge is regionally variable; cerebellum and cortex differ in important ways in their patterns of cellular and biochemical changes. Tracking these changes reveals an important though not exclusive role for the MAP kinase pathway. Our findings suggest brain responses to cytokine challenges are temporally and regionally specific and that both features are altered by the absence of ATM. This implies that management of the immune status of A-T patients might have significant clinical benefit.

Concurrent V(D)J recombination and DNA end instability increase interchromosomal trans -rearrangements in ATM-deficient thymocytes

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.

Ataxia Telangiectasia Mutated Kinase Controls Chronic Gammaherpesvirus Infection

Gammaherpesviruses, such as Epstein-Barr virus (EBV), are ubiquitous cancer-associated pathogens that interact with DNA damage response, a tumor suppressor network. Chronic gammaherpesvirus infection and pathogenesis in a DNA damage response-insufficient host are poorly understood. Ataxia-telangiectasia (A-T) is associated with insufficiency of ataxia-telangiectasia mutated (ATM), a critical DNA damage response kinase. A-T patients display a pattern of anti-EBV antibodies suggestive of poorly controlled EBV replication; however, parameters of chronic EBV infection and pathogenesis in the A-T population remain unclear. Here we demonstrate that chronic gammaherpesvirus infection is poorly controlled in an animal model of A-T. Intriguingly, in spite of a global increase in T cell activation and numbers in wild-type (wt) and ATM-deficient mice in response to mouse gammaherpesvirus 68 (MHV68) infection, the generation of an MHV68-specific immune response was altered in the absence of ATM. Our finding that ATM expression is necessary for an optimal adaptive immune response against gammaherpesvirus unveils an important connection between DNA damage response and immune control of chronic gammaherpesvirus infection, a connection that is likely to impact viral pathogenesis in an ATM-insufficient host.

Roles of ATM and ATR-Mediated DNA Damage Responses during Lytic BK Polyomavirus Infection

BK polyomavirus (BKPyV) is an emerging pathogen whose reactivation causes severe disease in transplant patients. Unfortunately, there is no specific anti-BKPyV treatment available, and host cell components that affect the infection outcome are not well characterized. In this report, we examined the relationship between BKPyV productive infection and the activation of the cellular DNA damage response (DDR) in natural host cells. Our results showed that both the ataxia-telangiectasia mutated (ATM)- and ATM and Rad-3-related (ATR)-mediated DDR were activated during BKPyV infection, accompanied by the accumulation of polyploid cells. We assessed the involvement of ATM and ATR during infection using small interfering RNA (siRNA) knockdowns. ATM knockdown did not significantly affect viral gene expression, but reduced BKPyV DNA replication and infectious progeny production. ATR knockdown had a slightly more dramatic effect on viral T antigen (TAg) and its modified forms, DNA replication, and progeny production. ATM and ATR double knockdown had an additive effect on DNA replication and resulted in a severe reduction in viral titer. While ATM mainly led to the activation of pChk2 and ATR was primarily responsible for the activation of pChk1, knockdown of all three major phosphatidylinositol 3-kinase-like kinases (ATM, ATR, and DNA-PKcs) did not abolish the activation of γH2AX during BKPyV infection. Finally, in the absence of ATM or ATR, BKPyV infection caused severe DNA damage and aberrant TAg staining patterns. These results indicate that induction of the DDR by BKPyV is critical for productive infection, and that one of the functions of the DDR is to minimize the DNA damage which is generated during BKPyV infection.

Drosophila ATM and ATR have distinct activities in the regulation of meiotic DNA damage and repair

Ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia-related (ATR) kinases are conserved regulators of cellular responses to double strand breaks (DSBs). During meiosis, however, the functions of these kinases in DSB repair and the deoxyribonucleic acid (DNA) damage checkpoint are unclear. In this paper, we show that ATM and ATR have unique roles in the repair of meiotic DSBs in Drosophila melanogaster. ATR mutant analysis indicated that it is required for checkpoint activity, whereas ATM may not be. Both kinases phosphorylate H2AV (γ-H2AV), and, using this as a reporter for ATM/ATR activity, we found that the DSB repair response is surprisingly dynamic at the site of DNA damage. γ-H2AV is continuously exchanged, requiring new phosphorylation at the break site until repair is completed. However, most surprising is that the number of γ-H2AV foci is dramatically increased in the absence of ATM, but not ATR, suggesting that the number of DSBs is increased. Thus, we conclude that ATM is primarily required for the meiotic DSB repair response, which includes functions in DNA damage repair and negative feedback control over the level of programmed DSBs during meiosis.

Loss of ATM positively regulates the expression of hypoxia inducible factor 1 (HIF-1) through oxidative stress: Role in the physiopathology of the disease

Ataxia Telangiectasia (AT) is an autosomal recessive disorder characterized by a wide variety of progressive clinical symptoms. This includes neuronal degeneration, oculocutaneous telangiectasias, diabetes mellitus, immunodeficiency, increased risk of cancer and sensitivity to ionizing radiation. The gene mutated in this disease, ATM (Ataxia Telangiectasia Mutated), encodes a protein kinase involved in DNA double strand breaks signalling and repair. ATM deficient cells also display an increase in oxidative stress, by poorly characterized mechanism(s), which clearly contributes to the neurodegenerative aspect of the disease. Despite these advances, the occurrence of the vascular abnormalities, glucose intolerance and insulin resistance remains poorly understood. In different cellular models where ATM expression was disrupted, we demonstrated that the absence of ATM leads to an increased expression of both subunits of the transcription factor Hypoxia Inducible Factor 1 (HIF-1). We also observed enhanced trans-activating functions of HIF-1. HIF-1 is the central regulator of responses to hypoxia which induces the transcription of genes involved in angiogenesis (e.g VEGF -Vascular Endothelial Growth Factor) and cellular metabolism (e.g GLUT-1). Interestingly, we demonstrated that ATM disruption positively regulates both expression and function of the basal glucose transporter GLUT-1 as well as the proangiogenic factor, VEGF. In addition, our results suggest that the absence of ATM increases HIF-1 proteins biosynthesis, and this effect is dependant on the oxidative stress existing in ATM deficient cells. Our compelling results highlight a new link between ATM deficiency and the clinical features of the disease and provide a molecular link between ATM down-regulation and the increase in tumor angiogenesis observed in human breast cancers.

Transcription-dependent Activation of Ataxia Telangiectasia Mutated Prevents DNA-dependent Protein Kinase-mediated Cell Death in Response to Topoisomerase I Poison

Camptothecin (CPT) is a topoisomerase I inhibitor, derivatives of which are being used for cancer chemotherapy. CPT-induced DNA double-strand breaks (DSBs) are considered a major cause of its tumoricidal activity, and it has been shown that CPT induces DNA damage signaling through the phosphatidylinositol 3-kinase-related kinases, including ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3-related), and DNA-PK (DNA-dependent protein kinase). In addition, CPT causes DNA strand breaks mediated by transcription, although the downstream signaling events are less well characterized. In this study, we show that CPT-induced activation of ATM requires transcription. Mechanistically, transcription inhibition suppressed CPT-dependent activation of ATM and blocked recruitment of the DNA damage mediator p53-binding protein 1 (53BP1) to DNA damage sites, whereas ATM inhibition abrogated CPT-induced G(1)/S and S phase checkpoints. Functional inactivation of ATM resulted in DNA replication-dependent hyperactivation of DNA-PK in CPT-treated cells and dramatic CPT hypersensitivity. On the other hand, simultaneous inhibition of ATM and DNA-PK partially restored CPT resistance, suggesting that activation of DNA-PK is proapoptotic in the absence of ATM. Correspondingly, comet assay and cell cycle synchronization experiments suggested that transcription collapse occurring as the result of CPT treatment are converted to frank double-strand breaks when ATM-deficient cells bypass the G(1)/S checkpoint. Thus, ATM suppresses DNA-PK-dependent cell death in response to topoisomerase poisons, a finding with potential clinical implications.

ATM and p53 are essential in the cell-cycle containment of DNA breaks during V(D)J recombination in vivo

V(D)J recombination is essential for the maturation of lymphocytes. Because of the involvement of cutting and joining DNA double strands, this recombination activity is strictly contained within the noncycling phases of the cell cycle. Such containment is crucial for the maintenance of genomic integrity. The ataxia telangiectasia mutated (ATM) gene is known to have a central role in sensing general DNA damage and mediating cell-cycle checkpoint. In this study, we investigated the role of ATM and its downstream targets in the cell-cycle control of V(D)J recombination in vivo. Our results revealed the persistence of double-strand breaks (DSBs) throughout the cell cycle in ATM(-/-) and p53(-/-) thymocytes, but the cell-cycle regulation of a V(D)J recombinase, Rag-2, was normal. The histone variant H2AX, which is phosphorylated during normal V(D)J recombination, was dispensable for containing DSBs. H2AX was still phosphorylated at V(D)J loci in the absence of ATM. Therefore, V(D)J recombination, a physiological DNA rearrangement process, activates the ATM/p53 pathway to contain DNA breaks within the noncycling cells and surprisingly this pathway is not important for containing Rag-2 activity. This study shows the dynamic multiple functions of ATM in maintaining genomic stability and preventing tumorigenesis in developing lymphocytes.

ATM Signaling Facilitates Repair of DNA Double-Strand Breaks Associated with Heterochromatin

Ataxia Telangiectasia Mutated (ATM) signaling is essential for the repair of a subset of DNA double-strand breaks (DSBs); however, its precise role is unclear. Here, we show that < or =25% of DSBs require ATM signaling for repair, and this percentage correlates with increased chromatin but not damage complexity. Importantly, we demonstrate that heterochromatic DSBs are generally repaired more slowly than euchromatic DSBs, and ATM signaling is specifically required for DSB repair within heterochromatin. Significantly, knockdown of the transcriptional repressor KAP-1, an ATM substrate, or the heterochromatin-building factors HP1 or HDAC1/2 alleviates the requirement for ATM in DSB repair. We propose that ATM signaling temporarily perturbs heterochromatin via KAP-1, which is critical for DSB repair/processing within otherwise compacted/inflexible chromatin. In support of this, ATM signaling alters KAP-1 affinity for chromatin enriched for heterochromatic factors. These data suggest that the importance of ATM signaling for DSB repair increases as the heterochromatic component of a genome expands.

ATM regulates the length of individual telomere tracts in Arabidopsis

Telomeres have the paradoxical ability of protecting linear chromosome ends from DNA damage sensors by using these same proteins as essential components of their maintenance machinery. We have previously shown that the absence of ataxia telangiectasia mutated (ATM), a central regulator of the DNA damage response, accelerates the onset of genome instability in telomerase-deficient Arabidopsis, without increasing the rate of bulk telomere shortening. Here, we examine individual telomere tracts through successive plant generations using both fluorescence situ in hybridization (FISH) and primer extension telomere repeat amplification (PETRA). Unexpectedly, we found that the onset of profound developmental defects and abundant end-to-end chromosome fusions in fifth generation (G(5)) atm tert mutants required the presence of only one critically shortened telomere. Parent progeny analysis revealed that the short telomere arose as a consequence of an unusually large telomere rapid deletion (TRD) event. The most dramatic TRD was detected in atm tert mutants that had undergone meiosis. Notably, in contrast to TRD, alternative lengthening of telomeres (ALT) was suppressed in the absence of ATM. Finally, we show that size differences between telomeres on homologous chromosome ends are greater for atm tert than tert plants. Altogether, these findings suggest a dual role for ATM in regulating telomere size by promoting elongation of short telomeres and by preventing the accumulation of cells that harbor large telomere deletions.

ATM deficiency impairs thymocyte maturation because of defective resolution of T cell receptor α locus coding end breaks

The ATM (ataxia telangiectasia mutated) protein plays a central role in sensing and responding to DNA double-strand breaks. Lymphoid cells are unique in undergoing physiologic double-strand breaks in the processes of Ig class switch recombination and T or B cell receptor V(D)J recombination, and a role for ATM in these processes has been suggested by clinical observations in ataxia telangiectasia patients as well as in engineered mice with mutations in the Atm gene. We demonstrate here a defect in thymocyte maturation in ATM-deficient mice that is associated with decreased efficiency in V-J rearrangement of the endogenous T cell receptor (TCR)alpha locus, accompanied by increased frequency of unresolved TCR Jalpha coding end breaks. We also demonstrate that a functionally rearranged TCRalphabeta transgene is sufficient to restore thymocyte maturation, whereas increased thymocyte survival by bcl-2 cannot improve TCRalpha recombination and T cell development. These data indicate a direct role for ATM in TCR gene recombination in vivo that is critical for surface TCR expression in CD4(+)CD8(+) cells and for efficient thymocyte selection. We propose a unified model for the two major clinical characteristics of ATM deficiency, defective T cell maturation and increased genomic instability, frequently affecting the TCRalpha locus. In the absence of ATM, delayed TCRalpha coding joint formation results both in a reduction of alphabeta TCR-expressing immature cells, leading to inefficient thymocyte selection, and in accumulation of unstable open chromosomal DNA breaks, predisposing to TCRalpha locus-associated chromosomal abnormalities.

Role of ATM in V(D)J recombination and cell cycle control (35.10)

Abstract V(D)J recombination is a process specific to developing B- and T- lymphocytes in which double strand breaks (DSBs) are introduced into germline DNA and upon repair generate rearranged gene segments. This recombination event is cell cycle regulated and is confined to the noncycling G0/G1 phase of cell cycle. The protein kinase Ataxia Telangiectasia Mutated (ATM) is a vital mediator in both the DNA damage response and cell cycle. ATM was found localized at V(D)J mediated break sites, suggesting an additional function for ATM in recombination. In the absence of ATM, mice develop thymic lymphomas with chromosomal translocations. Therefore, we hypothesize a role for ATM in the cell cycle control of V(D)J recombination. Thymic lymphomas from Atm null mice revealed cells harbored multiple chromosomal translocations near sites of recombination. Wildtype and Atm null thymocytes were separated into cell cycle fractions of G0/G1 and S/G2/M. V(D)J mediated DSBs, indicative of recombination, were observed at the T cell receptor α, δ, and β loci in both cell cycle fractions of Atm null thymocytes but were found only in the G0/G1 fraction of wildtype thymocytes. Together, these results indicate the involvement of ATM in the cell cycle control of V(D)J recombination. Loss of this control can lead to sustained DNA DSBs and translocations, which could contribute to tumorigenesis.

Single Chemical Modifications of the C-1027 Enediyne Core, a Radiomimetic Antitumor Drug, Affect Both Drug Potency and the Role of Ataxia-Telangiectasia Mutated in Cellular Responses to DNA Double-Strand Breaks

The radiomimetic enediyne C-1027 induces almost exclusively DNA double-strand breaks (DSB) and is extremely cytotoxic. Unique among radiomimetics, ataxia-telangiectasia mutated (ATM) is dispensable for cellular responses to C-1027-induced DNA damage. This study explores the biological activity of three recently bioengineered C-1027 analogues: 7''-desmethyl-C-1027 (desmethyl), 20'-deschloro-C-1027 (deschloro), and 22'-deshydroxy-C-1027 (deshydroxy). Each compound maintains the characteristic ability of radiomimetics to cleave DNA in cell-free systems, varying in activity from 2-fold (deschloro) to 55-fold (desmethyl) less than C-1027. The induction of cellular DNA breaks based on pulsed field gel electrophoresis, comet analysis, and gammaH2AX activation was in the same rank order as cell-free DNA break induction, although the amount of breaks induced by desmethyl is greatly reduced compared with the other analogues. Despite the disparity in inducing DNA DSBs, all of the analogues produced G2-M cell cycle arrest and activated DNA DSB damage response proteins, such as p53-Ser15 and Chk2-Thr68, at concentrations in concordance with their ability to inhibit cell growth. Interestingly, of the three analogues, only the desmethyl-induced DNA damage response was similar to C-1027, as it did not cause hypersensitive cell growth inhibition in the absence of ATM nor require the kinase to phosphorylate p53 or Chk2. These findings show that simple modifications of the chromophore of C-1027 can result in varied induction of, and cellular response to, DNA DSBs.

ATM and ATR promote Mre11 dependent restart of collapsed replication forks and prevent accumulation of DNA breaks

Ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia Rad3-related (ATR) and the Mre11/Rad50/Nbs1 complex ensure genome stability in response to DNA damage. However, their essential role in DNA metabolism remains unknown. Here we show that ATM and ATR prevent accumulation of DNA double-strand breaks (DSBs) during chromosomal replication. Replicating chromosomes accumulate DSBs in Xenopus laevis egg extracts depleted of ATM and ATR. Addition of ATM and ATR proteins to depleted extracts prevents DSB accumulation by promoting restart of collapsed replication forks that arise during DNA replication. We show that collapsed forks maintain MCM complex but lose Pol epsilon, and that Pol epsilon reloading requires ATM and ATR. Replication fork restart is abolished in Mre11 depleted extracts and is restored by supplementation with recombinant human Mre11/Rad50/Nbs1 complex. Using a novel fluorescence resonance energy transfer-based technique, we demonstrate that ATM and ATR induce Mre11/Rad50/Nbs1 complex redistribution to restarting forks. This study provides direct biochemical evidence that ATM and ATR prevent accumulation of chromosomal abnormalities by promoting Mre11/Rad50/Nbs1 dependent recovery of collapsed replication forks.

Rapid Activation of ATR by Ionizing Radiation Requires ATM and Mre11

The ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) protein kinases are crucial regulatory proteins in genotoxic stress response pathways that pause the cell cycle to permit DNA repair. Here we show that Chk1 phosphorylation in response to hydroxyurea and ultraviolet radiation is ATR-dependent and ATM- and Mre11-independent. In contrast, Chk1 phosphorylation in response to ionizing radiation (IR) is dependent on ATR, ATM, and Mre11. The ATR and ATM/Mre11 pathways are generally thought to be separate with ATM activation occurring early and ATR activation occurring as a late response to double strand breaks. However, we demonstrate that ATR is activated rapidly by IR, and ATM and Mre11 enhance ATR signaling. ATR-ATR-interacting protein recruitment to double strand breaks is less efficient in the absence of ATM and Mre11. Furthermore, IR-induced replication protein A foci formation is defective in ATM- and Mre11-deficient cells. Thus, ATM and Mre11 may stimulate the ATR signaling pathway by converting DNA damage generated by IR into structures that recruit and activate ATR.

The radiomimetic enediyne C-1027 induces unusual DNA damage responses to double-strand breaks.

Cells lacking the protein kinase ataxia telangiectasia mutated (ATM) have defective responses to DNA double-strand breaks (DSBs), including an inability to activate damage response proteins such as p53. However, we previously showed that cells lacking ATM robustly activate p53 in response to DNA strand breaks induced by the radiomimetic enediyne C-1027. To gain insight into the nature of C-1027-induced ATM-independent damage responses to DNA DSBs, we further examined the molecular mechanisms underlying the cellular response to this unique radiomimetic agent. Like ionizing radiation (IR) and other radiomimetics, breaks induced by C-1027 efficiently activate ATM by phosphorylation at Ser1981, yet unlike other radiomimetics and IR, DNA breaks induced by C-1027 result in normal phosphorylation of p53 and the cell cycle checkpoint kinases (Chk1 and Chk2) in the absence of ATM. In the presence of ATM, but under ATM and Rad3-related kinase (ATR) deficient conditions, C-1027 treatment resulted in a decrease in the level of Chk1 phosphorylation but not in the level of p53 and Chk2 phosphorylation. Only when cells were deficient in both ATM and ATR was there a reduction in the level of phosphorylation of each of these DNA damage response proteins. This reduction was also accompanied by an increased level of cell death in comparison to that of wild-type cells or cells lacking either ATM or ATR. Our findings demonstrate a unique cellular response to C-1027-induced DNA DSBs in that DNA damage response proteins are unaffected by the absence of ATM, as long as ATR is present.

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.