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  • The ATM kinase signaling induced by the low-energy β-particles emitted by (33)P is essential for the suppression of chromosome aberrations and is greater than that induce ... 21315088

    Ataxia-telangiectasia mutated (ATM) encodes a nuclear serine/threonine protein kinase whose activity is increased in cells exposed to low doses of ionizing radiation (IR). Here we examine ATM kinase activation in cells exposed to either (32)P- or (33)P-orthophosphate under conditions typically employed in metabolic labelling experiments. We calculate that the absorbed dose of IR delivered to a 5cm×5cm monolayer of cells incubated in 2ml media containing 1mCi of the high-energy (1.70MeV) β-particle emitter (32)P-orthophosphate for 30min is ∼1Gy IR. The absorbed dose of IR following an otherwise identical exposure to the low-energy (0.24MeV) β-particle emitter (33)P-orthophosphate is ∼0.18Gy IR. We show that low-energy β-particles emitted by (33)P induce a greater number of ionizing radiation-induced foci (IRIF) and greater ATM kinase signaling than energetic β-particles emitted by (32)P. Hence, we demonstrate that it is inappropriate to use (33)P-orthophosphate as a negative control for (32)P-orthophosphate in experiments investigating DNA damage responses to DNA double-strand breaks (DSBs). Significantly, we show that ATM accumulates in the chromatin fraction when ATM kinase activity is inhibited during exposure to either radionuclide. Finally, we also show that chromosome aberrations accumulate in cells when ATM kinase activity is inhibited during exposure to ∼0.36Gy β-particles emitted by (33)P. We therefore propose that direct cellular exposure to (33)P-orthophosphate is an excellent means to induce and label the IR-induced, ATM kinase-dependent phosphoproteome.Copyright © 2011 Elsevier B.V. All rights reserved.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • High expression of Wee1 is associated with poor disease-free survival in malignant melanoma: potential for targeted therapy. 22719872

    Notoriously resistant malignant melanoma is one of the most increasing forms of cancer worldwide; there is thus a precarious need for new treatment options. The Wee1 kinase is a major regulator of the G(2)/M checkpoint, and halts the cell cycle by adding a negative phosphorylation on CDK1 (Tyr15). Additionally, Wee1 has a function in safeguarding the genome integrity during DNA synthesis. To assess the role of Wee1 in development and progression of malignant melanoma we examined its expression in a panel of paraffin-embedded patient derived tissue of benign nevi and primary- and metastatic melanomas, as well as in agarose-embedded cultured melanocytes. We found that Wee1 expression increased in the direction of malignancy, and showed a strong, positive correlation with known biomarkers involved in cell cycle regulation: Cyclin A (pless than 0.0001), Ki67 (pless than 0.0001), Cyclin D3 (p = 0.001), p21(Cip1/WAF1) (p = 0.003), p53 (p = 0.025). Furthermore, high Wee1 expression was associated with thicker primary tumors (p = 0.001), ulceration (p = 0.005) and poor disease-free survival (p = 0.008). Transfections using siWee1 in metastatic melanoma cell lines; WM239(WTp53), WM45.1(MUTp53) and LOX(WTp53), further support our hypothesis of a tumor promoting role of Wee1 in melanomas. Whereas no effect was observed in LOX cells, transfection with siWee1 led to accumulation of cells in G(1)/S and S phase of the cell cycle in WM239 and WM45.1 cells, respectively. Both latter cell lines displayed DNA damage and induction of apoptosis, in the absence of Wee1, indicating that the effect of silencing Wee1 may not be solely dependent of the p53 status of the cells. Together these results reveal the importance of Wee1 as a prognostic biomarker in melanomas, and indicate a potential role for targeted therapy, alone or in combination with other agents.
    Document Type:
    Reference
    Product Catalog Number:
    05-636
    Product Catalog Name:
    Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301

  • Toxicity and genotoxicity of nano-SiO2 on human epithelial intestinal HT-29 cell line. 22378843

    Silica mesoporous nanoparticles have been recently selected for a wide range of applications from electronics to medicine due to their intrinsic properties. Among medical applications, drug delivery using SiO(2) nanoparticles by oral route is under study. Major benefits are expected including higher specificity and sensitivity together with side effect reduction. Since literature shows that very complex and unexpected interactions could occur between nanomaterials and biological systems, one critical issue is to control the nanoparticle cytotoxicity/genotoxicity for normal tissues and specially stomach and intestine when oral route is considered. The aim of the work is to study the cytotoxicity and genotoxicity of SiO(2) nanoparticles on HT29 human intestine cell line, using conventional and innovative methodologies, for measuring cell viability and proliferation, global metabolism, genotoxicity, and nanoparticles uptake. Core-dye doped SiO(2) nanoparticles of 25 and 100 nm were specifically synthesized to track nanoparticles incorporation by confocal and video microscopy. Besides conventional approaches (sulforhodamine B, flow cytometry, and γ-H2Ax foci), we have performed a real-time monitoring of cell proliferation using an impedance-based system which ensure no interference between measures and nanoparticles physicochemical characteristics. Overall, our results showed that SiO(2)-25nm and SiO(2)-100nm induced a rather limited cytotoxic and genotoxic effects on HT-29 cells after a 24 h exposure. However, regarding cell viability and genotoxicity, inverse dose-dependent relationships were observed for SiO(2)-100nm nanoparticles. In conclusion, it seems that the higher the dose of SiO(2)-100nm, the lower the cytotoxic/genotoxic effects, data that well illustrate the complexity in identifying and understanding the hazards of nanoparticles for human health.
    Document Type:
    Reference
    Product Catalog Number:
    05-636
    Product Catalog Name:
    Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301

  • Parp1-XRCC1 and the repair of DNA double strand breaks in mouse round spermatids. 19887075

    The repair of DNA double strand breaks (DSBs) in male germ cells is slower and differently regulated compared to that in somatic cells. Round spermatids show DSB repair and are radioresistant to apoptosis induction. Mutation induction studies using ionizing irradiation, indicated a high frequency of chromosome aberrations (CA) in the next generation. Since they are in a G1 comparable stage of the cell cycle, haploid spermatids are expected to repair DSBs by the non-homologous end-joining pathway (NHEJ). However, immunohistochemical evidence indicates that not all components of the classical NHEJ pathway are available since the presence of DNA-PKcs cannot be shown. Here, we demonstrate that round spermatids, as well as most other types of male germ cells express both Parp1 and XRCC1. Therefore, we have determined whether the alternative Parp1/XRCC1 dependent NHEJ pathway is active in these nuclei and also have tested for classical NHEJ activity by a genetic method. To evaluate DSB repair in SCID mice, deficient for DNA-PKcs, and to study the involvement of the Parp1/XRCC1 dependent NHEJ pathway in round spermatids, the loss of gamma-H2AX foci after irradiation has been determined in nucleus spreads of round spermatids of SCID mice and in nucleus spreads and histological sections of Parp1-inhibited mice and their respective controls. Results show that around half of the breaks in randomly selected round spermatids are repaired between 1 and 8h after irradiation. The repair of 16% of the induced DSBs requires DNA-PKcs and 21% Parp1. Foci numbers in the Parp1-inhibited testes tend to be higher in spermatids of all epithelial stages reaching significance in stages I-III which indicates an active Parp1/XRCC1 pathway in round spermatids and a decreased repair capacity in later round spermatid stages. In Parp1-inhibited SCID mice only 14.5% of the breaks were repaired 8h after irradiation indicating additivity of the two NHEJ pathways in round spermatids.
    Document Type:
    Reference
    Product Catalog Number:
    05-636
    Product Catalog Name:
    Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301

  • Metformin reduces endogenous reactive oxygen species and associated DNA damage. 22262811

    Pharmacoepidemiologic studies provide evidence that use of metformin, a drug commonly prescribed for type II diabetes, is associated with a substantial reduction in cancer risk. Experimental models show that metformin inhibits the growth of certain neoplasms by cell autonomous mechanisms such as activation of AMP kinase with secondary inhibition of protein synthesis or by an indirect mechanism involving reduction in gluconeogenesis leading to a decline in insulin levels and reduced proliferation of insulin-responsive cancers. Here, we show that metformin attenuates paraquat-induced elevations in reactive oxygen species (ROS), and related DNA damage and mutations, but has no effect on similar changes induced by H(2)0(2), indicating a reduction in endogenous ROS production. Importantly, metformin also inhibited Ras-induced ROS production and DNA damage. Our results reveal previously unrecognized inhibitory effects of metformin on ROS production and somatic cell mutation, providing a novel mechanism for the reduction in cancer risk reported to be associated with exposure to this drug.
    Document Type:
    Reference
    Product Catalog Number:
    05-636
    Product Catalog Name:
    Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301

  • Radiation Sensitivity and Tumor Susceptibility in ATM Phospho-Mutant ATF2 Mice. 20740050

    The transcription factor ATF2 was previously shown to be an ATM substrate. Upon phosphorylation by ATM, ATF2 exhibits a transcription-independent function in the DNA damage response through localization to DNA repair foci and control of cell cycle arrest. To assess the physiological significance of this phosphorylation, we generated ATF2 mutant mice in which the ATM phosphoacceptor sites (S472/S480) were mutated (ATF2(KI)). ATF2(KI) mice are more sensitive to ionizing radiation (IR) than wild-type (ATF2 (WT)) mice: following IR, ATF2(KI) mice exhibited higher levels of apoptosis in the intestinal crypt cells and impaired hepatic steatosis. Molecular analysis identified impaired activation of the cell cycle regulatory protein p21(Cip/Waf1) in cells and tissues of IR-treated ATF2(KI) mice, which was p53 independent. Analysis of tumor development in p53(KO) crossed with ATF2(KI) mice indicated a marked decrease in amount of time required for tumor development. Further, when subjected to two-stage skin carcinogenesis process, ATF2(KI) mice developed skin tumors faster and with higher incidence, which also progressed to the more malignant carcinomas, compared with the control mice. Using 3 mouse models, we establish the importance of ATF2 phosphorylation by ATM in the acute cellular response to DNA damage and maintenance of genomic stability.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Stage-Specific Modulation of Cortical Neuronal Development by Mmu-miR-134. 21228099

    To realize the potential of microRNAs (miRs) as fine-tuning regulators of embryonic neuronal differentiation, it is critical to define their developmental function. Mmu-miR-134 (miR-134) is a powerful inducer of pluripotent stem cell differentiation. However, its functional role during embryonic, neuronal development is unknown. We demonstrate that mature, miR-134 transcript levels elevate during embryonic, neuronal differentiation in vitro and in vivo. To define the developmental targets and function of miR-134, we identified multiple brain-expressed targets including the neural progenitor cell-enriched, bone morphogenetic protein (BMP) antagonist Chordin-like 1 (Chrdl-1) and the postmitotic, neuron-specific, microtubule-associated protein, Doublecortin (Dcx). We show that, through interaction with Dcx and/or Chrdl-1, miR-134 has stage-specific effects on cortical progenitors, migratory neurons, and differentiated neurons. In neural progenitors, miR-134 promotes cell proliferation and counteracts Chrdl-1-induced apoptosis and Dcx-induced differentiation in vitro. In neurons, miR-134 reduces cell migration in vitro and in vivo in a Dcx-dependent manner. In differentiating neurons, miR-134 modulates process outgrowth in response to exogenous BMP-4 in a noggin-reversible manner. Taken together, we present Dcx and Chrdl-1 as new regulatory targets of miR-134 during embryonic, mouse, cortical, and neuronal differentiation and show a novel and previously undiscovered role for miR-134 in the stage-specific modulation of cortical development.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • COA 105636

    Document Type:
    Certificate of Analysis
    Product Catalog Number:
    105636