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Title: Molecular Imaging of the ATM Kinase Activity

Purpose: Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including from DNA double-strand breaks. ATM activation results in the initiation of a complex cascade of events including DNA damage repair, cell cycle checkpoint control, and survival. We sought to create a bioluminescent reporter that dynamically and noninvasively measures ATM kinase activity in living cells and subjects. Methods and Materials: Using the split luciferase technology, we constructed a hybrid cDNA, ATM-reporter (ATMR), coding for a protein that quantitatively reports on changes in ATM kinase activity through changes in bioluminescence. Results: Treatment of ATMR-expressing cells with ATM inhibitors resulted in a dose-dependent increase in bioluminescence activity. In contrast, induction of ATM kinase activity upon irradiation resulted in a decrease in reporter activity that correlated with ATM and Chk2 activation by immunoblotting in a time-dependent fashion. Nuclear targeting improved ATMR sensitivity to both ATM inhibitors and radiation, whereas a mutant ATMR (lacking the target phosphorylation site) displayed a muted response. Treatment with ATM inhibitors and small interfering (si)RNA-targeted knockdown of ATM confirm the specificity of the reporter. Using reporter expressing xenografted tumors demonstrated the ability of ATMR to report in ATM activity in mouse models that correlatedmore » in a time-dependent fashion with changes in Chk2 activity. Conclusions: We describe the development and validation of a novel, specific, noninvasive bioluminescent reporter that enables monitoring of ATM activity in real time, in vitro and in vivo. Potential applications of this reporter include the identification and development of novel ATM inhibitors or ATM-interacting partners through high-throughput screens and in vivo pharmacokinetic/pharmacodynamic studies of ATM inhibitors in preclinical models.« less
Authors:
 [1] ;  [2] ;  [3] ;  [2] ;  [3] ;  [2] ;  [3] ;  [3]
  1. Department of Radiation Oncology, Ohio State University, Columbus, Ohio (United States)
  2. Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States)
  3. (United States)
Publication Date:
OSTI Identifier:
22267844
Resource Type:
Journal Article
Resource Relation:
Journal Name: International Journal of Radiation Oncology, Biology and Physics; Journal Volume: 86; Journal Issue: 5; Other Information: Copyright (c) 2013 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; 62 RADIOLOGY AND NUCLEAR MEDICINE; BIOLUMINESCENCE; CELL CYCLE; DNA; IN VITRO; IN VIVO; LUCIFERASE; MICE; NEOPLASMS; PHOSPHORYLATION; RNA; SERINE; STRAND BREAKS; THREONINE; TIME DEPENDENCE; VALIDATION