Article

Radiation therapy induces the DNA damage response in peripheral blood

Department of Radiation Oncology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Oncotarget (Impact Factor: 6.63). 06/2013; 4(8).
Source: PubMed

ABSTRACT Stereotactic body radiation therapy (SBRT) is a radiotherapy modality that delivers highly conformal, ablative doses to a well-defined target. Here, using a semiquantitative multiplexed assay to analyze ATM and H2AX phosphorylation, we show that ATM kinase activity in peripheral blood mononuclear cells is induced following SBRT. This observation of a systemic ATM kinase-dependent DNA damage response in the peripheral blood is unprecedented and promotes the use of ATM serine-1981 phosphorylation as a predictive biomarker for DNA damaging modalities and ATM inhibitors.

Download full-text

Full-text

Available from: John C Schmitz, Aug 13, 2014
0 Followers
 · 
89 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Pharmacologic inhibition of DNA repair may increase the efficacy of many cytotoxic cancer agents. Inhibitors of DNA repair enzymes including APE1, ATM, ATR, DNA-PK and PARP have been developed and the PARP inhibitor olaparib is the first-in-class approved in Europe and the USA for the treatment of advanced BRCA-mutated ovarian cancer. Sensitive pharmacodynamic (PD) biomarkers are needed to further evaluate the efficacy of inhibitors of DNA repair enzymes in clinical trials. ATM is a protein kinase that mediates cell-cycle checkpoint activation and DNA double-strand break repair. ATM kinase activation at DNA double-strand breaks (DSBs) is associated with intermolecular autophosphorylation on serine-1981. Exquisite sensitivity and high stoichiometry as well as facile extraction suggest that ATM serine-1981 phosphorylation may be a highly dynamic PD biomarker for both ATM kinase inhibitors and radiation- and chemotherapy-induced DSBs. Here we report the pre-clinical analytical validation and fit-for-purpose biomarker method validation of a quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX phosphorylation in human peripheral blood mononuclear cells (PBMCs). We explore the dynamics of these phosphorylations in PBMCs exposed to chemotherapeutic agents and DNA repair inhibitors in vitro, and show that ATM serine-1981 phosphorylation is increased in PBMCs in sarcoma patients treated with DNA damaging chemotherapy.
    2(5):542-554.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The aim of this study was to investigate impact of exogenous nitric oxide (NO) on generation of different types of DNA damages, their transformation, and specificity of DNA repair in cells treated with ionizing radiation (IR). Methods: levels of single-strand and double-strand breaks assessed in peripheral blood lymphocytes (PBL) isolated from healthy humans and treated in vitro with NO donor - S-nitrosoglutathione (GSNO) and IR. The rate of DNA repair estimated after 30 and 60 min of PBL treatment. The visualization and measuring the number of prompt and delayed DNA damages, including strand breaks, apurinic and thermolabile sites performed with single-cell gel electrophoresis. Results: IR caused dose-dependent generation of single strand breaks (SSBs), double strand breaks (DSBs), and heat-labile sites (HLS) in cell DNA. However, particularly destructive was combined treatment IR with GSNO as NO donor that leads to a significant increase of DNA damage and a dose-dependent inhibition of the DNA repair rate. Obtained data proofs the ability of NO to inhibit fast and slow stages of SSBs, DSBs, and HLS repair resulting in significant growth of genotoxic effect. DNA breaks generation from HLS is able to affect DSBs yields especially in cells with altered DNA repair. The process of DNA repair of delayed DSBs formed from HLS was quite different from removal of DNA damages occurring immediately after treatment and was characterized by IR dose dependent inhibition of DNA repair. Conclusion: High level of DNA strand breaks, that are generated after the combined treatment with NO and IR, are accumulated for quite a long time after exposure due to altered DNA repair, indicating the development of genetic instability and increase of carcinogenic risk for organism exposed to combination of harmful environmental factors.
    Experimental oncology 12/2013; 35(4):318-24.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Ionizing radiation increases cell mortality in a dose-dependent manner. Increases in DNA double strand breaks, γ-H2AX, p53 phophorylation, and protein levels of p53 and Bax also occur. We investigated the ability of ciprofloxacin (CIP), a widely prescribed antibiotic, to inhibit DNA damage induced by ionizing radiation. Human tumor TK6, NH32 (p53−/− of TK6) cells, and human normal peripheral blood mononuclear cells (PBMCs) were exposed to 2–8 Gy 60Co-γ-photon radiation. γ-H2AX (an indicator of DNA strand breaks), phosphorylated p53 (responsible for cell-cycle arrest), Bcl-2 (an apoptotic protein, and cell death were measured. Ionizing irradiation increased γ-H2AX amounts in TK6 cells (p53+/+) within 1 h in a radiation dose-dependent manner. CIP pretreatment and posttreatment effectively inhibited the increase in γ-H2AX. CIP pretreatment reduced Bcl-2 production but promoted p53 phosphorylation, caspase-3 activation and cell death. In NH32 cells, CIP failed to significantly inhibit the radiation-induced γ-H2AX increase, suggesting that CIP inhibition involves in p53-dependent mechanisms. In normal healthy human PBMCs, CIP failed to block the radiation-induced γ-H2AX increase but effectively increased Bcl-2 production, but blocked the phospho-p53 increase and subsequent cell death. CIP increased Gadd45α, and enhanced p21 protein 24 h postirradiation. Results suggest that CIP exerts its effect in TK6 cells by promoting p53 phosphorylation and inhibiting Bcl-2 production and in PBMCs by inhibiting p53 phosphorylation and increasing Bcl-2 production. Our data are the first to support the view that CIP may be effective to protect normal tissue cells from radiation injury, while enhancing cancer cell death in radiation therapy.
    Molecular and Cellular Biochemistry 08/2014; 393(1-2). DOI:10.1007/s11010-014-2053-z · 2.39 Impact Factor
Show more