Antioxidant N-acetyl cysteine reduces incidence and multiplicity of lymphoma in Atm deficient mice

Hereditary human disorder ataxia telangiectasia (AT) is characterized by an extremely high incidence of lymphoid malignancies, neuromotor dysfunction, immunodeficiency and radiosensitivity. Cells from AT patients show genetic instability and a continuous state of oxidative stress. We examined the effect of long-term dietary supplementation with the thiol-containing antioxidant, N-acetyl-L-cysteine (NAC), on survival and cancer formation in Atm (AT-mutated) deficient mice, used as an animal model of AT. NAC was chosen because it is well-tolerated in animals and humans. It can be used by the oral route and for long-term at high concentrations. In addition, NAC suppresses carcinogenesis-associated biological markers in Atm deficient mice, such as DNA deletions and oxidative DNA damage (R. Reliene, E. Fischer, R.H. Schiestl, Effect of N-acetyl cysteine on oxidative DNA damage and the frequency of DNA deletions in atm-deficient mice, Cancer Res. 64 (2004) 5148-5153). In this study, NAC significantly increased the lifespan and reduced both the incidence and multiplicity of lymphoma in Atm deficient mice. The life span increased from 50 to 68 weeks and the incidence of lymphoma decreased by two-fold (76.5% versus 37.5%). Moreover, in mice with lymphoma, multiplicity of tumors decreased from 4.6 to 2.8 tumors per mouse. Thus, dietary supplementation with NAC may turn out to be protective against lymphomagenesis in AT patients.     

RAG-mediated V(D)J recombination is not essential for tumorigenesis in Atm-deficient mice

Atm-deficient mice die of malignant thymic lymphomas characterized by translocations within the Tcr alpha/delta locus, suggesting that tumorigenesis is secondary to aberrant responses to double-stranded DNA (dsDNA) breaks that occur during RAG-dependent V(D)J recombination. We recently demonstrated that development of thymic lymphoma in Atm(-/-) mice was not prevented by loss of RAG-2. Thymic lymphomas that developed in Rag2(-/-) Atm(-/-) mice contained multiple chromosomal abnormalities, but none of these involved the Tcr alpha/delta locus. These findings indicated that tumorigenesis in Atm(-/-) mice is mediated by chromosomal translocations secondary to aberrant responses to dsDNA breaks and that V(D)J recombination is an important, but not essential, event in susceptibility. In contrast to these findings, it was recently reported that Rag1(-/-) Atm(-/-) mice do not develop thymic lymphomas, a finding that was interpreted as demonstrating a requirement for RAG-dependent recombination in the susceptibility to tumors in Atm-deficient mice. To test the possibility that RAG-1 and RAG-2 differ in their roles in tumorigenesis, we studied Rag1(-/-) Atm(-/-) mice in parallel to our previous Rag2(-/-) Atm(-/-) study. We found that thymic lymphomas occur at high frequency in Rag1(-/-) Atm(-/-) mice and resemble those that occur in Rag2(-/-) Atm(-/-) mice. These results indicate that both RAG-1 and RAG-2 are necessary for tumorigenesis involving translocation in the Tcr alpha/delta locus but that Atm deficiency leads to tumors through a broader RAG-independent predisposition to translocation, related to a generalized defect in dsDNA break repair.     

Tumor suppression by p53 in the absence of Atm

Oncogenes can induce p53 through a signaling pathway involving p19/Arf. It was recently proposed that oncogenes can also induce DNA damage, and this can induce p53 through the Atm DNA damage pathway. To assess the relative roles of Atm, Arf, and p53 in the suppression of Ras-driven tumors, we examined susceptibility to skin carcinogenesis in 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate (TPA)-treated Atm- and p53-deficient mice and compared these results to previous studies on Arf-deficient mice. Mice with epidermal-specific deletion of p53 showed increased papilloma number and progression to malignant invasive carcinomas compared with wild-type littermates. In contrast, Atm-deficient mice showed no increase in papilloma number, growth, or malignant progression. gamma-H2AX and p53 levels were increased in both Atm(+/+) and Atm(-/-) papillomas, whereas Arf(-/-) papillomas showed much lower p53 expression. Thus, although there is evidence of DNA damage, signaling through Arf seems to regulate p53 in these Ras-driven tumors. In spontaneous and radiation-induced lymphoma models, tumor latency was accelerated in Atm(-/-)p53(-/-) compound mutant mice compared with the single mutant Atm(-/-) or p53(-/-) mice, indicating cooperation between loss of Atm and loss of p53. Although p53-mediated apoptosis was impaired in irradiated Atm(-/-) lymphocytes, p53 loss was still selected for during lymphomagenesis in Atm(-/-) mice. In conclusion, in these models of oncogene- or DNA damage-induced tumors, p53 retains tumor suppressor activity in the absence of Atm.     

ATM controls c-Myc and DNA synthesis during postnatal thymocyte development through regulation of redox state

The oncoprotein c-Myc is essential for thymocyte development, and its dysregulation causes lymphoid malignancies. We have demonstrated previously that spontaneous DNA synthesis in Atm(-/-) thymocytes is markedly increased over that of Atm(+/+) thymocytes and that glucocorticoid dexamethasone suppresses thymocyte DNA synthesis and prevents the ultimate development of thymic lymphoma in Atm(-/-) mice. Recently, we reported that in Atm(-/-) thymic lymphoma cells c-Myc is overexpressed compared with the levels of c-Myc in primary thymocytes from wild-type or Atm(-/-) mice. In this study, we show that c-Myc expression progressively increases with age in primary thymocytes from Atm(-/-) mice and that the upregulation of c-Myc parallels the elevated DNA synthesis in the cells, suggesting that deregulation of c-Myc may drive the uncontrolled proliferation of thymocytes in Atm(-/-) mice. Here we also demonstrate that Atm(-/-) thymocytes exhibit increased levels of hydrogen peroxide, NF-E2-related factor (Nrf-2), peroxiredoxin-1, and intracellular glutathione relative to thymocytes from Atm(+/+) mice. Importantly, reduction of hydrogen peroxide by administration of the antioxidant N-acetylcysteine to Atm(-/-) mice attenuates the elevation of Nrf-2, c-Myc, and DNA synthesis in their thymocytes, suggesting that ATM may control c-Myc and DNA synthesis during postnatal thymocyte development by preventing accumulation of reactive oxygen species.     

Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells

Hematopoietic stem cells (HSCs) undergo self-renewing cell divisions and maintain blood production for their lifetime. Appropriate control of HSC self-renewal is crucial for the maintenance of hematopoietic homeostasis. Here we show that activation of p38 MAPK in response to increasing levels of reactive oxygen species (ROS) limits the lifespan of HSCs in vivo. In Atm(-/-) mice, elevation of ROS levels induces HSC-specific phosphorylation of p38 MAPK accompanied by a defect in the maintenance of HSC quiescence. Inhibition of p38 MAPK rescued ROS-induced defects in HSC repopulating capacity and in the maintenance of HSC quiescence, indicating that the ROS-p38 MAPK pathway contributes to exhaustion of the stem cell population. Furthermore, prolonged treatment with an antioxidant or an inhibitor of p38 MAPK extended the lifespan of HSCs from wild-type mice in serial transplantation experiments. These data show that inactivation of p38 MAPK protects HSCs against loss of self-renewal capacity. Our characterization of molecular mechanisms that limit HSC lifespan may lead to beneficial therapies for human disease.     

Loss of p53 Ser18 and Atm results in embryonic lethality without cooperation in tumorigenesis

Phosphorylation at murine Serine 18 (human Serine 15) is a critical regulatory process for the tumor suppressor function of p53. p53Ser18 residue is a substrate for ataxia-telangiectasia mutated (ATM) and ATM-related (ATR) protein kinases. Studies of mice with a germ-line mutation that replaces Ser18 with Ala (p53(S18A) mice) have demonstrated that loss of phosphorylation of p53Ser18 leads to the development of tumors, including lymphomas, fibrosarcomas, leukemia and leiomyosarcomas. The predominant lymphoma is B-cell lymphoma, which is in contrast to the lymphomas observed in Atm(-/-) animals. This observation and the fact that multiple kinases phosphorylate p53Ser18 suggest Atm-independent tumor suppressive functions of p53Ser18. Therefore, in order to examine p53Ser18 function in relationship to ATM, we analyzed the lifespan and tumorigenesis of mice with combined mutations in p53Ser18 and Atm. Surprisingly, we observed no cooperation in survival and tumorigenesis in compound p53(S18A) and Atm(-/-) animals. However, we observed embryonic lethality in the compound mutant animals. In addition, the homozygous p53Ser18 mutant allele impacted the weight of Atm(-/-) animals. These studies examine the genetic interaction of p53Ser18 and Atm in vivo. Furthermore, these studies demonstrate a role of p53Ser18 in regulating embryonic survival and motor coordination.     

Atm deficiency affects both apoptosis and proliferation to augment Myc-induced lymphomagenesis

Myc oncoproteins are commonly activated in malignancies and are sufficient to provoke many types of cancer. However, the critical mechanisms by which Myc contributes to malignant transformation are not clear. DNA damage seems to be an important initiating event in tumorigenesis. Here, we show that although Myc does not directly induce double-stranded DNA breaks, it does augment activation of the Atm/p53 DNA damage response pathway, suggesting that Atm may function as a guardian against Myc-induced transformation. Indeed, we show that Atm loss augments Myc-induced lymphomagenesis and impairs Myc-induced apoptosis, which normally harnesses Myc-driven tumorigenesis. Surprisingly, Atm loss also augments the proliferative response induced by Myc, and this augmentation is associated with enhanced suppression of the expression of the cyclin-dependent kinase inhibitor p27(Kip1). Therefore, regulation of cell proliferation and p27(Kip1) seems to be a contributing mechanism by which Atm holds tumor formation in check.     

A role for Xrcc2 in the early stages of mouse development

Xrcc2 is one of a family of five Rad51-like genes with important roles in the repair of DNA damage by homologous recombination (HR) in mammals. We have shown previously that loss of Xrcc2 in mice results in severe but variable developmental defects and embryonic lethality, potentially linked to excessive apoptosis. To look at the causes of lethality, and possibly to allow Xrcc2-/- mice to survive to birth, we have produced double knockout mice deficient in either the p53 oncoprotein or Ataxia telangiectasia mutated (Atm). Overall we show that the excessive apoptosis observed in Xrcc2-/- embryos is p53-dependent, and that loss of p53 can restore growth capacity to Xrcc2-/- fibroblasts in culture, but that it cannot rescue the embryonic lethality. Additionally, although the Xrcc2-/- Trp53-/- embryos show a near-normal morphology they remain relatively small in size. Loss of Atm in an Xrcc2-/- embryo has little effect, suggesting that response to loss of HR capacity is not mediated through the Atm kinase in the early stages of mouse development. Further, as seen by reduced expression of the early developmental marker, Delta-like1, the normal developmental programme is perturbed in Xrcc2-/- embryonic tissues, particularly during neurogenesis and somitogenesis. Taken together our data suggest that the accumulation of spontaneous damage in HR-deficient embryos has severe consequences for the development and survival of mammals due to the unregulated loss of cells important to the developmental programme.     

Ataxia-telangiectasia mutated is not required for p53 induction and apoptosis in irradiated epithelial tissues

The ataxia-telangiectasia mutated (Atm) protein kinase is a central regulator of the cellular response to DNA damage. Although Atm can regulate p53, it is not known if this Atm function varies between tissues. Previous studies showed that the induction of p53 and apoptosis by whole-body ionizing radiation varies greatly between tissue and tumor types, so here we asked if Atm also had a tissue-specific role in the ionizing radiation response. Irradiated Atm-null mice showed impaired p53 induction and apoptosis in thymus, spleen, and brain. In contrast, radiation-induced p53, apoptosis, phosphorylation of Chk2, and G(2)-M cell cycle arrest were slightly delayed in Atm(-/-) epithelial cells of the small intestine but reached wild-type levels by 4 h. Radiation-induced p53 and apoptosis in Atm(-/-) hair follicle epithelial cells were not impaired at any of the time points examined. Thus, Atm is essential for radiation-induced apoptosis in lymphoid tissues but is largely dispensable in epithelial cells. This indicates that marked differences in DNA damage signaling pathways exist between tissues, which could explain some of the tissue-specific phenotypes, especially tumor suppression, associated with Atm deficiency.     

Stem cell defects in ATM-deficient undifferentiated spermatogonia through DNA damage-induced cell-cycle arrest

Mammalian spermatogenesis is maintained by stem cell capacity within undifferentiated spermatogonial subpopulation. Here, using a combination of surface markers, we describe a purification method for undifferentiated spermatogonia. Flow cytometric analysis revealed that this population is composed of Plzf-positive cells and exhibits quiescence and the side population phenotype, fulfilling general stem cell criteria. We then applied this method to analyze undifferentiated spermatogonia and stem cell activity of Atm(-/-) mice. Atm(-/-) testis shows progressive depletion of undifferentiated spermatogonia accompanied by cell-cycle arrest. In Atm(-/-) undifferentiated spermatogonia, a self-renewal defect was observed in vitro and in vivo. Accumulation of DNA damage and activation of the p19(Arf)-p53-p21(Cip1/Waf1) pathway were observed in Atm(-/-) undifferentiated spermatogonia. Moreover, suppression of p21(Cip1/Waf1) in an Atm(-/-) background restored transplantation ability of undifferentiated spermatogonia, indicating that ATM plays an essential role in maintenance of undifferentiated spermatogonia and their stem cell capacity by suppressing DNA damage-induced cell-cycle arrest.     

Activation of AMP-activated protein kinase in cerebella of Atm-/- mice is attributable to accumulation of reactive oxygen species

Ataxia telangiectasia (A-T) is an inherited disease, the most prominent feature of which is ataxia caused by degeneration of cerebellar neurons and synapses. The mechanisms underlying A-T neurodegeneration are still unclear, and many factors are likely to be involved. AMP-activated protein kinase (AMPK) is a sensor of energy balance, and research on its function in neural cells has gained momentum in the last decade. The dual roles of AMPK in neuroprotection and neurodegeneration are complex, and they need to be identified and characterized. Using an Atm (ataxia telangiectasia mutated) gene deficient mouse model, we showed here that: (a) upregulation of AMPK phosphorylation and elevation of reactive oxygen species (ROS) coordinately occur in the cerebella of Atm-/- mice; (b) hydrogen peroxide induces AMPK phosphorylation in primary mouse cerebellar astrocytes in an Atm-independent manner; (c) administration of the novel antioxidant monosodium luminol (MSL) to Atm-/- mice attenuates the upregulation of both phosphorylated-AMPK (p-AMPK) and ROS, and corrects the neuromotor deficits in these animals. Together, our results suggest that oxidative activation of AMPK in the cerebellum may contribute to the neurodegeneration in Atm-/- mice, and that ROS and AMPK signaling pathways are promising therapeutic targets for treatment of A-T and other neurodegenerative diseases.     

The ROS scavenger, NAC, regulates hepatic Vα14iNKT cells signaling during Fas mAb-dependent fulminant liver failure

Uncontrolled systemic activation of the immune system is an early initiating event that leads to development of acute fulminant liver failure (FLF) in mice after treatment with agonistic Fas mAb. In this study, we demonstrate that treatment of mice with N-acetylcysteine (NAC), an ROS scavenger and glutathione (GSH) precursor, almost completely abolished Fas mAb-induced FLF through suppression of Vα14iNKT cell activation, IFN-γ signaling, apoptosis and nitrotyrosine formation in liver. In addition, enrichment of the liver with GSH due to Vα14iNKT cells deficiency, induced an anti-inflammatory response in the liver of Jα18(-/-) mice that inhibited apoptosis, nitrotyrosine formation, IFN-γ signaling and effector functions. In summary, we propose a novel and previously unrecognized pro-inflammatory and pro-apoptotic role for endogenous ROS in stimulating Th1 signaling in Vα14iNKT cells to promote the development of FLF. Therefore, our study provides critical new insights into how NAC, a ROS scavenger, regulates Th1 signaling in intrahepatic Vα14iNKT cells to impact inflammatory and pathological responses.     

Redundant and nonredundant functions of ATM and H2AX in αβ T-lineage lymphocytes

The ataxia telangiectasia mutated (ATM) kinase and H2AX histone tumor suppressor proteins are each critical for maintenance of cellular genomic stability and suppression of lymphomas harboring clonal translocations. ATM is the predominant kinase that phosphorylates H2AX in chromatin around DNA double-strand breaks, including along lymphocyte Ag receptor loci cleaved during V(D)J recombination. However, combined germline inactivation of Atm and H2ax in mice causes early embryonic lethality associated with substantial cellular genomic instability, indicating that ATM and H2AX exhibit nonredundant functions in embryonic cells. To evaluate potential nonredundant roles of ATM and H2AX in somatic cells, we generated and analyzed Atm-deficient mice with conditional deletion of H2ax in αβ T-lineage lymphocytes. Combined Atm/H2ax inactivation starting in early-stage CD4(-)/CD8(-) thymocytes resulted in lower numbers of later-stage CD4(+)/CD8(+) thymocytes, but led to no discernible V(D)J recombination defect in G1 phase cells beyond that observed in Atm-deficient cells. H2ax deletion in Atm-deficient thymocytes also did not affect the incidence or mortality of mice from thymic lymphomas with clonal chromosome 14 (TCRα/δ) translocations. Yet, in vitro-stimulated Atm/H2ax-deficient splenic αβ T cells exhibited a higher frequency of genomic instability, including radial chromosome translocations and TCRβ translocations, compared with cells lacking Atm or H2ax. Collectively, our data demonstrate that both redundant and nonredundant functions of ATM and H2AX are required for normal recombination of TCR loci, proliferative expansion of developing thymocytes, and maintenance of genomic stability in cycling αβ T-lineage cells.     

Mutations that affect meiosis in male mice influence the dynamics of the mid-preleptotene and bouquet stages

Meiosis pairs and segregates homologous chromosomes and thereby forms haploid germ cells to compensate the genome doubling at fertilization. Homologue pairing in many eukaryotic species depends on formation of DNA double strand breaks (DSBs) during early prophase I when telomeres begin to cluster at the nuclear periphery (bouquet stage). By fluorescence in situ hybridization criteria, we observe that mid-preleptotene and bouquet stage frequencies are altered in male mice deficient for proteins required for recombination, ubiquitin conjugation and telomere length control. The generally low frequencies of mid-preleptotene spermatocytes were significantly increased in male mice lacking recombination proteins SPO11, MEI1, MLH1, KU80, ubiquitin conjugating enzyme HR6B, and in mice with only one copy of the telomere length regulator Terf1. The bouquet stage was significantly enriched in Atm(-/-), Spo11(-/-), Mei1(m1Jcs/m1Jcs), Mlh1(-/-), Terf1(+/-) and Hr6b(-/-) spermatogenesis, but not in mice lacking recombination proteins DMC1 and HOP2, the non-homologous end-joining DNA repair factor KU80 and the ATM downstream effector GADD45a. Mice defective in spermiogenesis (Tnp1(-/-), Gmcl1(-/-), Asm(-/-)) showed wild-type mid-preleptotene and bouquet frequencies. A low frequency of bouquet spermatocytes in Spo11(-/-)Atm(-/-) spermatogenesis suggests that DSBs contribute to the Atm(-/-)-correlated bouquet stage exit defect. Insignificant changes of bouquet frequencies in mice with defects in early stages of DSB repair (Dmc1(-/-), Hop2(-/-)) suggest that there is an ATM-specific influence on bouquet stage duration. Altogether, it appears that several pathways influence telomere dynamics in mammalian meiosis.     

Synthetic lethality between mutation in Atm and DNA-PK(cs) during murine embryogenesis

The gene product mutated in ataxia telangiectasia, ATM, is a ubiquitously expressed 370 kDa protein kinase that is a key mediator of the cellular response to DNA damage [1]. ATM-deficient cells are radiosensitive and show impaired cell cycle arrest and increased chromosome breaks in response to ionizing radiation. ATM is a member of the phosphatidylinositol-3-kinase (PI3K)-related protein kinase superfamily, which includes the catalytic subunit of DNA-dependent protein kinase (DNA-PK(cs)) and ATR [2]. DNA-PK is a 470 kDa protein kinase that is required for proper end-to-end rejoining of DNA double-strand breaks [3]. Prkdc(scid/scid) mice have a homozygous mutation in the gene encoding DNA-PK(cs) and, like Atm(-/-) mice, are viable and radiosensitive [4-8]. To determine if Atm and DNA-PK(cs) show genetic interaction, we attempted to generate mice deficient in both gene products. However, no scid/scid Atm(-/-) pups were recovered from scid/scid Atm(+/-) intercrosses. Developmental arrest of scid/scid Atm(-/-) embryos occurred around E7.5, a developmental stage when embryonic cells are hypersensitive to DNA damage [9]. This reveals synthetic lethality between mutations in Atm and DNA-PK and suggests that Atm and DNA-PK have complementary functions that are essential for development.     

Caspase-2 deficiency prevents programmed germ cell death resulting from cytokine insufficiency but not meiotic defects caused by loss of ataxia telangiectasia-mutated (Atm) gene function

It is well established that programmed cell death claims up to two-thirds of the oocytes produced during gametogenesis in the developing fetal ovaries. However, the mechanisms underlying prenatal germ cell loss in females remain poorly understood. Herein we report that caspase-11 null female mice are born with a reduced number of oocyte-containing primordial follicles. This phenotype is likely due to failed cytokine processing known to occur in caspase-11 mutants since neonatal female mice lacking both interleukin (IL)-1alpha and IL-1beta also exhibit a reduced endowment of primordial follicles. In addition, germ cell death in wild-type fetal ovaries cultured ex vivo is suppressed by either cytokine, likely via ligand activation of type 1 IL-1 receptors expressed in fetal germ cells. Normal oocyte endowment can be restored in caspase-11 null female mice by simultaneous inactivation of the gene encoding the cell death executioner enzyme, caspase-2. However, caspase-2 deficiency cannot overcome gametogenic failure resulting from meiotic recombination defects in ataxia telangiectasia-mutated (Atm) null female mice. Thus, genetically distinct mechanisms exist for developmental deletion of oocytes via programmed cell death, one of which probably functions as a meiotic quality-control checkpoint that cannot be overridden.     

Prdx1 deficiency in mice promotes tissue specific loss of heterozygosity mediated by deficiency in DNA repair and increased oxidative stress

The loss of the H(2)O(2) scavenger protein encoded by Prdx1 in mice leads to an elevation of reactive oxygen species (ROS) and tumorigenesis of different tissues. Loss of heterozygosity (LOH) mutations could initiate tumorigenesis through loss of tumor suppressor gene function in heterozygous somatic cells. A connection between the severity of ROS and the frequency of LOH mutations in vivo has not been established. Therefore, in this study, we characterized in vivo LOH in ear fibroblasts and splenic T cells of 3-4 month old Prdx1 deficient mice. We found that the loss of Prdx1 significantly elevates ROS amounts in T cells and fibroblasts. The basal amounts of ROS were higher in fibroblasts than in T cells, probably due to a less robust Prdx1 peroxidase activity in the former. Using Aprt as a LOH reporter, we observed an elevation in LOH mutation frequency in fibroblasts, but not in T cells, of Prdx1(-/-) mice compared to Prdx1(+/+) mice. The majority of the LOH mutations in both cell types were derived from mitotic recombination (MR) events. Interestingly, Mlh1, which is known to suppress MR between divergent sequences, was found to be significantly down-regulated in fibroblasts of Prdx1(-/-) mice. Therefore, the combination of elevated ROS amounts and down-regulation of Mlh1 may have contributed to the elevation of MR in fibroblasts of Prdx1(-/-) mice. We conclude that each tissue may have a distinct mechanism through which Prdx1 deficiency promotes tumorigenesis.