These documents contain iTerm(s) sense. You can select the species or the number of abstracts to display per page.
Sterigmatocystin (ST), which is commonly detected in food and feed commodities, is a mutagenic and carcinogenic mycotoxin that has been recognized as a possible human carcinogen. Our previous study showed that ST can induce G2 phase arrest in GES-1 cells in vitro and that the MAPK and PI3K signaling pathways are involved in the ST-induced G2 arrest. It is now widely accepted that DNA damage plays a critical role in the regulation of cell cycle arrest and apoptosis. In response to DNA damage, a complex signaling network is activated in eukaryotic cells to trigger cell cycle arrest and facilitate DNA repair. To further explore the molecular mechanism through which ST induces G2 arrest, the current study was designed to precisely dissect the role of DNA damage and the DNA damage sensor ataxia telangiectasia-mutated (Atm)/p53-dependent pathway in the ST-induced G2 arrest in GES-1 cells. Using the comet assay, we determined that ST induces DNA damage, as evidenced by the formation of DNA comet tails, in GES-1 cells. We also found that ST induces the activation of Atm and its downstream molecules, Chk2 and p53, in GES-1 cells. The Atm pharmacological inhibitor caffeine was found to effectively inhibit the activation of the Atm-dependent pathways and to rescue the ST-induced G2 arrest in GES-1 cells, which indicating its Atm-dependent characteristic. Moreover, the silencing of the p53 expression with siRNA effectively attenuated the ST-induced G2 arrest in GES-1 cells. We also found that ST induces apoptosis in GES-1 cells. Thus, our results show that the ST-induced DNA damage activates the Atm/53-dependent signaling pathway, which contributes to the induction of G2 arrest in GES-1 cells.
Ataxia telangiectasia (AT) is a human genetic disease characterized by radiation sensitivity, impaired neuronal development and predisposition to cancer. Using a genetically defined model cell system consisting of cells expressing a kinase dead or a kinase proficient Atm gene product, we previously reported systemic alterations in major metabolic pathways that translate at the gene expression, protein and small molecule metabolite levels. Here, we report ionizing radiation induced stress response signaling arising from perturbations in the Atm gene, by employing a functional proteomics approach. Functional pathway analysis shows robust translational and post-translational responses under Atm proficient conditions, which include enrichment of proteins in the Ephrin receptor and axonal guidance signaling pathways. These molecular networks offer a hypothesis generating function for further investigations of cellular stress responses.
Gliomas are resistant to radiation therapy, as well as to TNF? induced killing. Radiation-induced TNF? triggers Nuclear factor ?B (NF?B)-mediated radioresistance. As inhibition of NF?B activation sensitizes glioma cells to TNF?-induced apoptosis, we investigated whether TNF? modulates the responsiveness of glioma cells to ionizing radiation-mimetic Neocarzinostatin (NCS). TNF? enhanced the ability of NCS to induce glioma cell apoptosis. NCS-mediated death involved caspase-9 activation, reduction of mitochondrial copy number and lactate production. Death was concurrent with NF?B, Akt and Erk activation. Abrogation of Akt and NF?B activation further potentiated the death inducing ability of NCS in TNF? cotreated cells. NCS-induced p53 expression was accompanied by increase in TP53-induced glycolysis and apoptosis regulator (TIGAR) levels and Atm phosphorylation. siRNA-mediated knockdown of TIGAR abrogated NCS-induced apoptosis. While DN-I?B abrogated NCS-induced TIGAR both in the presence and absence of TNF?, TIGAR had no effect on NF?B activation. Transfection with TIGAR mutant (i) decreased apoptosis and ?H2AX foci formation (ii) decreased p53 (iii) elevated ROS and (iv) increased Akt/Erk activation in cells cotreated with NCS and TNF?. Heightened TIGAR expression was observed in GBM tumors. While NCS induced Atm phosphorylation in a NF?B independent manner, Atm inhibition abrogated TIGAR and NF?B activation. Metabolic gene profiling indicated that TNF? affects NCS-mediated regulation of several genes associated with glycolysis. The existence of Atm-NF?B axis that regulate metabolic modeler TIGAR to overcome prosurvival response in NCS and TNF? cotreated cells, suggests mechanisms through which inflammation could affect resistance and adaptation to radiomimetics despite concurrent induction of death.
Both radiotherapy and most effective chemotherapeutic agents induce different types of DNA damage. Here we show that tungstate modulates cell response to DNA damaging agents. Cells treated with tungstate were more sensitive to etoposide, phleomycin and ionizing radiation (IR), all of which induce DNA double-strand breaks (DSBs). Tungstate also modulated the activation of the central DSB signalling kinase, Atm, in response to these agents. These effects required the functionality of the Mre11-Nbs1-Rad50 (MRN) complex and were mimicked by the inhibition of PP2A phosphatase. Therefore, tungstate may have adjuvant activity when combined with DNA-damaging agents in the treatment of several malignancies.
Telomeres distinguish chromosome ends from double-strand breaks (DSBs) and prevent chromosome fusion. However, telomeres can also interfere with DNA repair, as shown by a deficiency in nonhomologous end joining (NHEJ) and an increase in large deletions at telomeric DSBs. The sensitivity of telomeric regions to DSBs is important in the cellular response to ionizing radiation and oncogene-induced replication stress, either by preventing cell division in normal cells, or by promoting chromosome instability in cancer cells. We have previously proposed that the telomeric protein TRF2 causes the sensitivity of telomeric regions to DSBs, either through its inhibition of Atm, or by promoting the processing of DSBs as though they are telomeres, which is independent of Atm. Our current study addresses the mechanism responsible for the deficiency in repair of DSBs near telomeres by combining assays for large deletions, NHEJ, small deletions, and gross chromosome rearrangements (GCRs) to compare the types of events resulting from DSBs at interstitial and telomeric DSBs. Our results confirm the sensitivity of telomeric regions to DSBs by demonstrating that the frequency of GCRs is greatly increased at DSBs near telomeres and that the role of Atm in DSB repair is very different at interstitial and telomeric DSBs. Unlike at interstitial DSBs, a deficiency in Atm decreases NHEJ and small deletions at telomeric DSBs, while it increases large deletions. These results strongly suggest that Atm is functional near telomeres and is involved in end protection at telomeric DSBs, but is not required for the extensive resection at telomeric DSBs. The results support our model in which the deficiency in DSB repair near telomeres is a result of Atm-independent processing of DSBs as though they are telomeres, leading to extensive resection, telomere loss, and GCRs involving alternative NHEJ.
Homology-directed repair (HDR) is a critical pathway for the repair of DNA double-strand breaks (DSBs) in mammalian cells. Efficient HDR is thought to be crucial for maintenance of genomic integrity during organismal development and tumor suppression. However, most mammalian HDR studies have focused on transformed and immortalized cell lines. We report here the generation of a Direct Repeat (DR)-GFP reporter-based mouse model to study HDR in primary cell types derived from diverse lineages. Embryonic and adult fibroblasts from these mice as well as cells derived from mammary epithelium, ovary, and neonatal brain were observed to undergo HDR at I-SceI endonuclease-induced DSBs at similar frequencies. When the DR-GFP reporter was crossed into mice carrying a hypomorphic mutation in the breast cancer susceptibility gene Brca1, a significant reduction in HDR was detected, showing that BRCA1 is critical for HDR in somatic cell types. Consistent with an HDR defect, Brca1 mutant mice are highly sensitive to the cross-linking agent mitomycin C. By contrast, loss of the DSB signaling ataxia telangiectasia-mutated (Atm) kinase did not significantly alter HDR levels, indicating that Atm is dispensable for HDR. Notably, chemical inhibition of Atm interfered with HDR. The DR-GFP mouse provides a powerful tool for dissecting the genetic requirements of HDR in a diverse array of somatic cell types in a normal, nontransformed cellular milieu.
Exposure of proliferating cells to genotoxic stresses activates a cascade of signaling events termed the DNA damage response (DDR). The DDR preserves genetic stability by detecting DNA lesions, activating cell cycle checkpoints and promoting DNA damage repair. The phosphoinositide 3-kinase-related kinases (PIKKs) ataxia telangiectasia-mutated (Atm), Atm and Rad 3-related kinase (ATR) and DNA-dependent protein kinase (DNA-PK) are crucial for sensing lesions and signal transduction. The checkpoint kinase 1 (CHK1) is a traditional ATR target involved in DDR and normal cell cycle progression and represents a pharmacological target for anticancer regimens. This study employed cell lines stably depleted for CHK1, Atm or both for dissecting cross-talk and compensatory effects on G(2)/M checkpoint in response to ionizing radiation (IR). We show that a 90% depletion of CHK1 renders cells radiosensitive without abrogating their IR-mediated G(2)/M checkpoint arrest. Atm phosphorylation is enhanced in CHK1-deficient cells compared with their wild-type counterparts. This correlates with lower nuclear abundance of the PP2A catalytic subunit in CHK1-depleted cells. Stable depletion of CHK1 in an Atm-deficient background showed only a 50% reduction from wild-type CHK1 protein expression levels and resulted in an additive attenuation of the G(2)/M checkpoint response compared with the individual knockdowns. Atm inhibition and 90% CHK1 depletion abrogated the early G(2)/M checkpoint and precluded the cells from mounting an efficient compensatory response to IR at later time points. Our data indicates that dual targeting of Atm and CHK1 functionalities disrupts the compensatory response to DNA damage and could be exploited for developing efficient anti-neoplastic treatments.
Little is known of the regulation of IL-23 secretion in dendritic cells (DC) despite its importance for human Th17 responses. In this study, we show for first time, to our knowledge, that the Ataxia telangiectasia mutated (Atm) pathway, involved in DNA damage sensing, acts as an IL-23 repressor. Inhibition of Atm with the highly selective antagonist KU55933 markedly increased IL-23 secretion in human monocyte-derived DC and freshly isolated myeloid DC. In contrast, inhibiting the closely related mammalian target of rapamycin had no effect on IL-23. Priming naive CD4(+) T cells with Atm-inhibited DC increased Th17 responses over and above those obtained with mature DC. Although Atm blockade increased the abundance of p19, p35, and p40 mRNA, IL-12p70 secretion was unaffected. To further examine a role for Atm in IL-23 regulation, we exposed DC to low doses of ionizing radiation. Exposure of DC to x-rays resulted in Atm phosphorylation and a corresponding depression of IL-23. Importantly, Atm inhibition with KU55933 prevented radiation-induced Atm phosphorylation and abrogated the capacity of x-rays to suppress IL-23. To explore how Atm repressed IL-23, we examined a role for endoplasmic reticulum stress responses by measuring generation of the spliced form of X-box protein-1, a key endoplasmic reticulum stress transcription factor. Inhibition of Atm increased the abundance of X-box protein-1 mRNA, and this was followed 3 h later by increased peak p19 transcription and IL-23 release. In summary, Atm activation or inhibition, respectively, inhibited or augmented IL-23 release. This novel role of the Atm pathway represents a new therapeutic target in autoimmunity and vaccine development.
Ataxia-telangiectasia (A-T) is an autosomal recessive neurodegenerative disorder characterized by radiosensitivity, genomic instability, and predisposition to cancer. A-T is caused by biallelic mutations in the ataxia-telangiectasia mutated (Atm) gene, but heterozygous carriers, though apparently healthy, are believed to be at increased risk for cancer and more sensitive to ionizing radiation than the general population. Despite progress in functional and sequencing-based assays, no straightforward, rapid, and inexpensive test is available for the identification of A-T homozygotes and heterozygotes, which is essential for diagnosis, genetic counseling, and carrier prediction. The oncosuppressor p53 prevents genomic instability and centrosomal amplification. During mitosis, p53 localizes at the centrosome in an Atm-dependent manner. We capitalized on the latter finding and established a simple, fast, minimally invasive, reliable, and inexpensive test to determine mutant Atm zygosity. The percentage of mitotic lymphoblasts or PBMCs bearing p53 centrosomal localization clearly discriminated among healthy donors (>75%), A-T heterozygotes (40%-56%), and A-T homozygotes (<30%). The test is specific for A-T, independent of the type of Atm mutations, and recognized tumor-associated Atm polymorphisms. In a preliminary study, our test confirmed that Atm is a breast cancer susceptibility gene. These data open the possibility of cost-effective, early diagnosis of A-T homozygotes and large-scale screenings for heterozygotes.