title: Longitudinal bioluminescence imaging of the dynamics of Doxorubicin induced apoptosis.
abstract: OBJECTIVE
  Most chemotherapy agents cause tumor cell death primarily by the induction of apoptosis. The ability to noninvasively image apoptosis in vivo could dramatically benefit pre-clinical and clinical evaluation of chemotherapeutics targeting the apoptotic pathway. This study aims to visualize the dynamics of apoptotic process with temporal bioluminescence imaging (BLI) using an apoptosis specific bioluminescence reporter gene.
  
  
  METHODS
  Both UM-SCC-22B human head and neck squamous carcinoma cells and 4T1 murine breast cancer cells were genetically modified with a caspase-3 specific cyclic firefly luciferase reporter gene (pcFluc-DEVD). Apoptosis induced by different concentrations of doxorubicin in the transfected cells was evaluated by both annexin V staining and BLI. Longitudinal BLI was performed in xenografted tumor models at different time points after doxorubicin or Doxil treatment, to evaluate apoptosis. After imaging, DNA fragmentation in apoptotic cells was assessed in frozen tumor sections using TUNEL staining.
  
  
  RESULTS
  Dose- and time-dependent apoptosis induced by doxorubicin in pcFluc-DEVD transfected UM-SCC-22B and 4T1 cells was visualized and quantified by BLI. Caspase-3 activation was confirmed by both caspase activity assay and Glo(TM) luciferase assay. One dose of doxorubicin treatment induced a dramatic increase in BLI intensity as early as 24 h after treatment in 22B-pcFluc-DEVD xenografted tumors. Sustained signal increase was observed for the first 3 days and the fluorescent signal from ex vivo TUNEL staining was consistent with BLI imaging results. Long-term imaging revealed that BLI signal consistently increased and reached a maximum at around day 12 after the treatment with one dose of Doxil.
  
  
  CONCLUSIONS
  BLI of apoptosis with pcFluc-DEVD as a reporter gene facilitates the determination of kinetics of the apoptotic process in a real-time manner, which provides a unique tool for drug development and therapy response monitoring.
authors.affiliation: Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda, MD 20892, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Niu
authors.forename: Lei
authors.initials: L
authors.lastname: Zhu
authors.forename: Don N
authors.initials: DN
authors.lastname: Ho
authors.forename: Fan
authors.initials: F
authors.lastname: Zhang
authors.forename: Haokao
authors.initials: H
authors.lastname: Gao
authors.forename: Qimeng
authors.initials: Q
authors.lastname: Quan
authors.forename: Naoki
authors.initials: N
authors.lastname: Hida
authors.forename: Takeaki
authors.initials: T
authors.lastname: Ozawa
authors.forename: Gang
authors.initials: G
authors.lastname: Liu
authors.forename: Xiaoyuan
authors.initials: X
authors.lastname: Chen
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000032:analysis; D000970:Antineoplastic Agents / Q000008:administration & dosage* / Q000494:pharmacology; D017209:Apoptosis; D053148:Caspase 3 / Q000032:analysis*; D045744:Cell Line, Tumor; D053938:DNA Fragmentation; D003952:Diagnostic Imaging / Q000379:methods*; D004195:Disease Models, Animal; D004317:Doxorubicin / Q000008:administration & dosage* / Q000494:pharmacology; D017930:Genes, Reporter; D006801:Humans; D020287:In Situ Nick-End Labeling; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods; D051379:Mice; D009369:Neoplasms / Q000175:diagnosis* / Q000188:drug therapy*; D013997:Time Factors
chemical_list: D017304:Annexin A5; D000970:Antineoplastic Agents; D004317:Doxorubicin; D049409:Luciferases, Firefly; D053148:Caspase 3
keywords: apoptosis; bioluminescence imaging; caspase-3.; cyclic firefly luciferase; doxorubicin