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      An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells

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          Abstract

          After a DNA damage signal multiple polymers of ADP ribose attached to poly(ADP) ribose (PAR) polymerases (PARPs) are broken down by the enzyme poly(ADP) ribose glycohydrolase (PARG). Inhibition of PARG leads to a failure of DNA repair and small molecule inhibition of PARG has been a goal for many years. To determine whether biochemical inhibitors of PARG are active in cells we have designed an immunofluorescence assay to detect nuclear PAR after DNA damage. This 384-well assay is suitable for medium throughput high-content screening and can detect cell-permeable inhibitors of PARG from nM to µM potency. In addition, the assay has been shown to work in murine cells and in a variety of human cancer cells. Furthermore, the assay is suitable for detecting the DNA damage response induced by treatment with temozolomide and methylmethane sulfonate (MMS). Lastly, the assay has been shown to be robust over a period of several years.

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          Most cited references22

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          Identification and characterization of a mammalian 39-kDa poly(ADP-ribose) glycohydrolase.

          ADP-ribosylation is a post-translational modification resulting from transfer of the ADP-ribose moiety of NAD to protein. Mammalian cells contain mono-ADP-ribosyltransferases that catalyze the formation of ADP-ribose-(arginine) protein, which can be cleaved by a 39-kDa ADP-ribose-(arginine) protein hydrolase (ARH1), resulting in release of free ADP-ribose and regeneration of unmodified protein. Enzymes involved in poly(ADP-ribosylation) participate in several critical physiological processes, including DNA repair, cellular differentiation, and carcinogenesis. Multiple poly(ADP-ribose) polymerases have been identified in the human genome, but there is only one known poly(ADP-ribose) glycohydrolase (PARG), a 111-kDa protein that degrades the (ADP-ribose) polymer to ADP-ribose. We report here the identification of an ARH1-like protein, termed poly(ADP-ribose) hydrolase or ARH3, which exhibited PARG activity, generating ADP-ribose from poly-(ADP-ribose), but did not hydrolyze ADP-ribose-arginine, -cysteine, -diphthamide, or -asparagine bonds. The 39-kDa ARH3 shares amino acid sequence identity with both ARH1 and the catalytic domain of PARG. ARH3 activity, like that of ARH1, was enhanced by Mg(2+). Critical vicinal acidic amino acids in ARH3, identified by mutagenesis (Asp(77) and Asp(78)), are located in a region similar to that required for activity in ARH1 but different from the location of the critical vicinal glutamates in the PARG catalytic site. All findings are consistent with the conclusion that ARH3 has PARG activity but is structurally unrelated to PARG.
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            Preclinical selection of a novel poly(ADP-ribose) polymerase inhibitor for clinical trial.

            Poly(ADP-ribose) polymerase (PARP)-1 (EC 2.4.2.30) is a nuclear enzyme that promotes the base excision repair of DNA breaks. Inhibition of PARP-1 enhances the efficacy of DNA alkylating agents, topoisomerase I poisons, and ionizing radiation. Our aim was to identify a PARP inhibitor for clinical trial from a panel of 42 potent PARP inhibitors (K(i), 1.4-15.1 nmol/L) based on the quinazolinone, benzimidazole, tricyclic benzimidazole, tricyclic indole, and tricyclic indole-1-one core structures. We evaluated chemosensitization of temozolomide and topotecan using LoVo and SW620 human colorectal cells; in vitro radiosensitization was measured using LoVo cells, and the enhancement of antitumor activity of temozolomide was evaluated in mice bearing SW620 xenografts. Excellent chemopotentiation and radiopotentiation were observed in vitro, with 17 of the compounds causing a greater temozolomide and topotecan sensitization than the benchmark inhibitor AG14361 and 10 compounds were more potent radiosensitizers than AG14361. In tumor-bearing mice, none of the compounds were toxic when given alone, and the antitumor activity of the PARP inhibitor-temozolomide combinations was unrelated to toxicity. Compounds that were more potent chemosensitizers in vivo than AG14361 were also more potent in vitro, validating in vitro assays as a prescreen. These studies have identified a compound, AG14447, as a PARP inhibitor with outstanding in vivo chemosensitization potency at tolerable doses, which is at least 10 times more potent than the initial lead, AG14361. The phosphate salt of AG14447 (AG014699), which has improved aqueous solubility, has been selected for clinical trial.
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              Targeting DNA repair pathways for cancer treatment: what's new?

              Disruptions in DNA repair pathways predispose cells to accumulating DNA damage. A growing body of evidence indicates that tumors accumulate progressively more mutations in DNA repair proteins as cancers progress. DNA repair mechanisms greatly affect the response to cytotoxic treatments, so understanding those mechanisms and finding ways to turn dysregulated repair processes against themselves to induce tumor death is the goal of all DNA repair inhibition efforts. Inhibition may be direct or indirect. This burgeoning field of research is replete with promise and challenge, as more intricacies of each repair pathway are discovered. In an era of increasing concern about healthcare costs, use of DNA repair inhibitors can prove to be highly effective stewardship of R&D resources and patient expenses.
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                Author and article information

                Journal
                F1000Res
                F1000Res
                F1000Research
                F1000Research
                F1000Research (London, UK )
                2046-1402
                25 April 2016
                2016
                : 5
                : 736
                Affiliations
                [1 ]Drug Discovery Unit, Cancer Research UK Manchester Institute, University of Manchester, Manchester, UK
                [2 ]Oncology iMED, AstraZeneca Pharmaceuticals, Macclesfield, UK
                [1 ]Department of Molecular Genetics, Erasmus University Medical Center, Rotterdam, Netherlands
                [2 ]Department of Pathology, Erasmus University Medical Center, Rotterdam, Netherlands
                [1 ]Department of Cancer Genetics and Epigenetics, Beckman Research Institute, City of Hope Medical Center, Duarte, CA, USA
                Author notes

                D.J., S.D., N.H., E.F., L.G., P.K., K.S. and K.E. designed and conducted the biological experiments. D.J. and S.D. conceptualized the experiments and I.W., M. O’C and D.O. provided strategic direction. D.J. prepared the manuscript.

                Competing interests: There are no competing financial interests to declare. Stephen Durant, Kay Eckersley, Kerry Shea, and Mark O’Connor were all employees of AstraZeneca PLC at the time experiments took place.

                Competing interests: No competing interests were disclosed.

                Competing interests: No competing interests were disclosed.

                Article
                10.12688/f1000research.8463.1
                4995692
                a20a2cb6-b4ea-49a5-bf51-4a34b35903e7
                Copyright: © 2016 James DI et al.

                This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 18 April 2016
                Funding
                Funded by: Cancer Research UK
                Award ID: C480/A1141
                Award ID: C5759/A17098
                This work was funded by Cancer Research UK (Grant numbers C480/A1141 and C5759/A17098).
                The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Method Article
                Articles
                Cell Signaling
                Medical Genetics
                Structure: Replication & Repair

                parg,parp,olaparib,dna damage response,base excision repair,mms,adp ribosylation

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