Transcriptional downregulation of ATM by EGF is defective in ataxia-telangiectasia cells expressing mutant protein

There is evidence that ATM plays a wider role in intracellular signalling in addition to DNA damage recognition and cell cycle control, In this report we show that activation of the EGF receptor is defective in ataxia-telangiectasia (A-T) cells and that sustained stimulation of cells with EGF downregulates ATM protein in control cells but not in A-T cells expressing mutant protein, Concomitant with the downregulation of ATM, DNA-binding activity of the transcription factor Spl decreased in controls after EGF treatment but increased from a lower basal level in A-T cells to that in untreated control cells, Mutation in two Spl consensus sequences in the ATM promoter reduced markedly the capacity of the promoter to support luciferase activity in a reporter assay. Overexpression of anti-sense ATM cDNA in control cells decreased the;basal level of Spl, which in turn was increased by subsequent treatment of cells with EGF, similar to that observed in,A-T cells. On the other hand full-length ATM cDNA increased the basal level of Spl binding in A-T cells, and in response to EGF Spl binding decreased, confirming that this is an ATR I-dependent process. Contrary to that observed in control cells there was no radiation-induced change in ATM protein in EGF-treated A-T cells and likewise no alteration in Spl binding activity. The results demonstrate that EGF-induced downregulation of ATM (mutant) protein in A-T cells is defective and this appears to be due to less efficient EGFR activation and abnormal Spl regulation.

Ataxia-telangiectasia: chronic activation of damage-responsive functions is reduced by alpha-lipoic acid

Cells from patients with the genetic disorder ataxia-telangiectasia (A-T) are hypersensitive to ionizing radiation and radiomimetic agents, both of which generate reactive oxygen species capable of causing oxidative damage to DNA and other macromolecules. We describe in A-T cells constitutive activation of pathways that normally respond to genotoxic stress, Basal levels of p53 and p21(WAF1/CIP1), phosphorylation on serine 15 of p53, and the Tyr15-phosphorylated form of cdc2 are chronically elevated in these cells. Treatment of A-T cells with the antioxidant alpha -lipoic acid significantly reduced the levels of these proteins, pointing to the involvement of reactive oxygen species in their chronic activation. These findings suggest that the absence of functional ATM results in a mild but continuous state of oxidative stress, which could account for several features of the pleiotropic phenotype of A-T.

Ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3 related kinase mediate phosphorylation of Brca1 at distinct and overlapping sites - In vivo assessment using phospho-specific antibodies

Recent studies have provided evidence that breast cancer susceptibility gene products (Brca1 and Brca2) suppress cancer, at least in part, by participating in DNA damage signaling and DNA repair. Brca1 is hyperphosphorylated in response to DNA damage and co-localizes with Rad51, a protein involved in homologous-recombination, and Nbs1·Mre11·Rad50, a complex required for both homologous-recombination and nonhomologous end joining repair of damaged DNA. Here, we report that there is a qualitative difference in the phosphorylation states of Brca1 between ionizing radiation (IR) and UV radiation. Brca1 is phosphorylated at Ser-1423 and Ser-1524 after IR and UV; however, Ser-1387 is specifically phosphorylated after IR, and Ser-1457 is predominantly phosphorylated after UV. These results suggest that different types of DNA-damaging agents might signal to Brca1 in different ways. We also provide evidence that the rapid phosphorylation of Brca1 at Ser-1423 and Ser-1524 after IR (but not after UV) is largely ataxia telangiectasia mutated (ATM) kinase-dependent. The overexpression of catalytically inactive ATM and Rad3 related (ATR) kinase inhibited the UV-induced phosphorylation of Brca1 at these sites, indicating that ATR controls Brca1 phosphorylation in vivo after the exposure of cells to UV light. Moreover, ATR associates with Brca1; ATR and Brca1 foci co-localize both in cells synchronized in S phase and after exposure of cells to DNA-damaging agents. ATR can itself phosphorylate the region of Brca1 phosphorylated by ATM (Ser-Gln cluster in the C terminus of Brca1, amino acids 1241-1530). However, there are additional uncharacterized ATR phosphorylation site(s) between residues 521 and 757 of Brca1. Taken together, our results support a model in which ATM and ATR act in parallel but somewhat overlapping pathways of DNA damage signaling but respond primarily to different types of DNA lesion.

Ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3 related kinase mediate phosphorylation of Brca1 at distinct and overlapping sites - In vivo assessment using phospho-specific antibodies

Recent studies have provided evidence that breast cancer susceptibility gene products (Brca1 and Brca2) suppress cancer, at least in part, by participating in DNA damage signaling and DNA repair. Brca1 is hyperphosphorylated in response to DNA damage and co-localizes with Rad51, a protein involved in homologous-recombination, and Nbs1.Mre11.Rad50, a complex required for both homologous-recombination and nonhomologous end joining repair of damaged DNA. Here, we report that there is a qualitative difference in the phosphorylation states of Brca1 between ionizing radiation (IR) and UV radiation. Brca1 is phosphorylated at Ser-1423 and Ser-1524 after IR and W; however, Ser-1387 is specifically phosphorylated after IR, and Ser-1457 is predominantly phosphorylated after W. These results suggest that different types of DNA-damaging agents might signal to Brca1 in different ways. We also provide evidence that the rapid phosphorylation of Brca1 at Ser-1423 and Ser-1524 after IR (but not after W) is largely ataxia telangiectasia mutated (ATM) kinase-dependent. The overexpression of catalytically inactive ATM and Rad3 related (ATR) kinase inhibited the UV-induced phosphorylation of Brca1 at these sites, indicating that ATR controls Brca1 phosphorylation in vivo after the exposure of cells to UV light. Moreover, ATR associates with Brca1; ATR and Brca1 foci co-localize both in cells synchronized in S phase and after exposure of cells to DNA-damaging agents. ATR can itself phosphorylate the region of Brca1 phosphorylated by ATM (Ser-Gln cluster in the C terminus of Brca1, amino acids 1241-1530), However, there are additional uncharacterized ATR phosphorylation site(s) between residues 521 and 757 of Brca1, Taken together, our results support a model in which ATM and ATR act in parallel but somewhat overlapping pathways of DNA damage signaling but respond primarily to different types of DNA lesion.

Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation of Chk1 on Ser-317 in response to ionizing radiation

In mammals, the ATM (ataxia-telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases function as critical regulators of the cellular DNA damage response. The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2. In mammalian cells, evidence has been presented that Chk1 is devoted to the ATR signaling pathway and is modified by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions primarily through ATM in response to ionizing radiation (IR), suggesting that Chk2 and Chk1 might have evolved to channel the DNA damage signal from ATM and ATR, respectively. We demonstrate here that the ATR-Chk1 and ATM-Chk2 pathways are not parallel branches of the DNA damage response pathway but instead show a high degree of cross-talk and connectivity. ATM does in fact signal to Chk1 in response to IR. Phosphorylation of Chk1 on Ser-317 in response to IR is ATM-dependent. We also show that functional NBS1 is required for phosphorylation of Chk1, indicating that NES1 might facilitate the access of Chk1 to ATM at the sites of DNA damage. Abrogation of Chk1 expression by RNA interference resulted in defects in IR-induced S and G2/M phase checkpoints; however, the overexpression of phosphorylation site mutant (S317A, S345A or S317A/S345A double mutant) Chk1 failed to interfere with these checkpoints. Surprisingly, the kinase-dead Chk1 (D130A) also failed to abrogate the S and G2 checkpoint through any obvious dominant negative effect toward endogenous Chk1. Therefore, further studies will be required to assess the contribution made by phosphorylation events to Chk1 regulation. Overall, the data presented in the study challenge the model in which Chk1 only functions downstream from ATR and indicate that ATM does signal to Chk1. In addition, this study also demonstrates that Chk1 is essential for IR-induced inhibition of DNA synthesis and the G2/M checkpoint.

ATP activates ataxia-telangiectasia mutated (ATM) in vitro - Importance of autophosphorylation

Ataxia-telangiectasia Mutated (ATM), mutated in the human disorder ataxia-telangiectasia, is rapidly activated by DNA double strand breaks. The mechanism of activation remains unresolved, and it is uncertain whether autophosphorylation contributes to activation. We describe an in vitro immunoprecipitation system demonstrating activation of ATM kinase from unirradiated extracts by preincubation with ATP. Activation is both time- and ATP concentration-dependent, other nucleotides fail to activate ATM, and DNA is not required. ATP activation is specific for ATM since it is not observed with kinase-dead ATM, it requires Mn2+, and it is inhibited by wortmannin. Exposure of activated ATM to phosphatase abrogates activity, and repeat cycles of ATP and phosphatase treatment reveal a requirement for autophosphorylation in the activation process. Phosphopeptide mapping revealed similarities between the patterns of autophosphorylation for irradiated and ATP-treated ATM. Caffeine inhibited ATM kinase activity for substrates but did not interfere with ATM autophosphorylation. ATP failed to activate either A-T and rad3-related protein (ATR) or DNA-dependent protein kinase under these conditions, supporting the specificity for ATM. These data demonstrate that ATP can specifically induce activation of ATM by a mechanism involving autophosphorylation. The relationship of this activation to DNA damage activation remains unclear but represents a useful model for understanding in vivo activation.

Ataxia telangiectasia mutated (ATM) inhibition transforms human mammary gland epithelial cells.

Carriers of mutations in the cell cycle checkpoint protein kinase ataxia telangiectasia mutated (ATM), which represent 1-2% of the general population, have an increased risk of breast cancer. However, experimental evidence that ATM deficiency contributes to human breast carcinogenesis is lacking. We report here that in MCF-10A and MCF-12A cells, which are well established normal human mammary gland epithelial cell models, partial or almost complete stable ATM silencing or pharmacological inhibition resulted in cellular transformation, genomic instability, and formation of dysplastic lesions in NOD/SCID mice. These effects did not require the activity of exogenous DNA-damaging agents and were preceded by an unsuspected and striking increase in cell proliferation also observed in primary human mammary gland epithelial cells. Increased proliferation correlated with a dramatic, transient, and proteasome-dependent reduction of p21(WAF1/CIP1) and p27(KIP1) protein levels, whereas little or no effect was observed on p21(WAF1/CIP1) or p27(KIP1) mRNAs. p21(WAF1/CIP1) silencing also increased MCF-10A cell proliferation, thus identifying p21(WAF1/CIP1) down-regulation as a mediator of the proliferative effect of ATM inhibition. Our findings provide the first experimental evidence that ATM is a human breast tumor suppressor. In addition, they mirror the sensitivity of ATM tumor suppressor function and unveil a new mechanism by which ATM might prevent human breast tumorigenesis, namely a direct inhibitory effect on the basal proliferation of normal mammary epithelial cells.

Il cane come modello animale spontaneo di patologie neoplastiche dell'uomo: ene Ataxia-Telangiectasia Mutated (ATM) Nnella predisposizione al cancro ed importanza dei riarrangimanti genici dei geni Ig/TCR per la diagnosi e prognosi delle malattie linfoproliferative

“Naturally occurring cancers in pet dogs and humans share many features, including histological appearance, tumour genetics, molecular targets, biological behaviour and response to conventional therapies. Studying dogs with cancer is likely to provide a valuable perspective that is distinct from that generated by the study of human or rodent cancers alone. The value of this opportunity has been increasingly recognized in the field of cancer research for the identification of cancer-associated genes, the study of environmental risk factors, understanding tumour biology and progression, and, perhaps most importantly, the evaluation and development of novel cancer therapeutics”.(Paoloni and Khanna, 2008) In last years, the author has investigated some molecular features of cancer in dogs. The Thesis is articulated in two main sections. In section 1, the preliminary results of a research project aimed at investigating the role of somatic mutations of Ataxia-Telangiectasia mutated (ATM) gene in predisposing to cancer in boxer dogs, are presented. The canine boxer breed may be considered an unique opportunity to disclose the role of ATM somatic mutation since boxer dogs are known to be dramatically susceptible to cancer and since they may be considered a closed gene pool. Furthermore, dogs share with human the some environment. Overall, the abovementioned features could be considered extremely useful for our purposes. In the section 2, the results of our studies aimed at setting up accurate and sensitive molecular assays for diagnosing and assessing minimal residual disease in lymphoproliferative disorders of dogs, are presented. The results of those molecular assay may be directly translated in the field of Veterinary practice as well as the may be used to improve our objective evaluation of new investigational drugs effectiveness in canine cancer trials.

EGF sensitizes cells to ionizing radiation by down-regulating protein mutated in ataxia-telangiectasia

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

Epidermal growth factor sensitizes cells to ionizing radiation by down-regulating protein mutated in ataxia-telangiectasia

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

Oxidative stress is responsible for deficient survival and dendritogenesis in Purkinje neurons from ataxia-telangiectasia mutated mutant mice

Atm gene-disrupted mice recapitulate the majority of characteristics observed in patients with the genetic disorder ataxia-telangiectasia (A-T). However, although they exhibit defects in neuromotor function and a distinct neurological phenotype, they do not show the progressive neurodegeneration seen in human patients, but there is evidence that ataxia-telangiectasia mutated ( Atm)-deficient animals have elevated levels of oxidized macromolecules and some neuropathology. We report here that in vitro survival of cerebellar Purkinje cells from both Atm knock-out and Atm knock-in mice was significantly reduced compared with their wild-type littermates. Although most of the Purkinje neurons from wild-type mice exhibited extensive dendritic elongation and branching under these conditions, most neurons from Atm-deficient mice had dramatically reduced dendritic branching. An antioxidant ( isoindoline nitroxide) prevented Purkinje cell death in Atm-deficient mice and enhanced dendritogenesis to wild-type levels. Furthermore, administration of the antioxidant throughout pregnancy had a small enhancing effect on Purkinje neuron survival in Atm gene-disrupted animals and protected against oxidative stress in older animals. These data provide strong evidence for a defect in the cerebellum of Atm-deficient mice and suggest that oxidative stress contributes to this phenotype.

Pleiotropic defects in ataxia-telangiectasia protein-deficient mice

We have generated a mouse model for ataxia-telangiectasia by using gene targeting to generate mice that do not express the Atm protein. Atm-deficient mice are retarded in growth, do not produce mature sperm, and exhibit severe defects in T cell maturation while going on to develop thymomas. Atm-deficient fibroblasts grow poorly in culture and display a high level of double-stranded chromosome breaks. Atm-deficient thymocytes undergo spontaneous apoptosis in vitro significantly more than controls. Atm-deficient mice then exhibit many of the same symptoms found in ataxia-telangiectasia patients and in cells derived from them. Furthermore, we demonstrate that the Atm protein exists as two discrete molecular species, and that loss of one or of both of these can lead to the development of the disease.

Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro

The protein kinase Chk2, the mammalian homolog of the budding yeast Rad53 and fission yeast Cds1 checkpoint kinases, is phosphorylated and activated in response to DNA damage by ionizing radiation (IR), UV irradiation, and replication blocks by hydroxyurea (HU). Phosphorylation and activation of Chk2 are ataxia telangiectasia-mutated (ATM) dependent in response to IR, whereas Chk2 phosphorylation is ATM-independent when cells are exposed to UV or HU. Here we show that in vitro, ATM phosphorylates the Ser-Gln/Thr-Gln (SQ/TQ) cluster domain (SCD) on Chk2, which contains seven SQ/TQ motifs, and Thr68 is the major in vitro phosphorylation site by ATM. ATM- and Rad3-related also phosphorylates Thr68 in addition to Thr26 and Ser50, which are not phosphorylated to a significant extent by ATM in vitro. In vivo, Thr68 is phosphorylated in an ATM-dependent manner in response to IR, but not in response to UV or HU. Substitution of Thr68 with Ala reduced the extent of phosphorylation and activation of Chk2 in response to IR, and mutation of all seven SQ/TQ motifs blocked all phosphorylation and activation of Chk2 after IR. These results suggest that in vivo, Chk2 is directly phosphorylated by ATM in response to IR and that Chk2 is regulated by phosphorylation of the SCD.