title: Molecular imaging of the ATM kinase activity.
abstract: OBJECTIVE
  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
  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 correlated 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.
authors.affiliation: Department of Radiation Oncology, Ohio State University, Columbus, Ohio, USA.
authors.forename: Terence M
authors.initials: TM
authors.lastname: Williams
authors.forename: Shyam
authors.initials: S
authors.lastname: Nyati
authors.forename: Brian D
authors.initials: BD
authors.lastname: Ross
authors.forename: Alnawaz
authors.initials: A
authors.lastname: Rehemtulla
mesh_terms: D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D044482:Benzeneacetamides / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D002467:Cell Nucleus / Q000201:enzymology; D064447:Checkpoint Kinase 2; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000528:radiation effects; D005260:Female; D017930:Genes, Reporter / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods*; D051379:Mice; D008819:Mice, Nude; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D034741:RNA, Small Interfering / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013890:Thiourea / Q000031:analogs & derivatives / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects
chemical_list: D044482:Benzeneacetamides; C512273:CGK 733; D018797:Cell Cycle Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D002110:Caffeine; D008156:Luciferases; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; C578634:CHEK2 protein, human; C578633:Chek2 protein, mouse; D017346:Protein Serine-Threonine Kinases; D013890:Thiourea