title: Novel Bioluminescent Activatable Reporter for Src Tyrosine Kinase Activity in Living Mice.
abstract: Aberrant activation of the Src kinase is implicated in the development of a variety of human malignancies. However, it is almost impossible to monitor Src activity in an in vivo setting with current biochemical techniques. To facilitate the noninvasive investigation of the activity of Src kinase both in vitro and in vivo, we developed a genetically engineered, activatable bioluminescent reporter using split-luciferase complementation. The bioluminescence of this reporter can be used as a surrogate for Src activity in real time. This hybrid luciferase reporter was constructed by sandwiching a Src-dependent conformationally responsive unit (SH2 domain-Srcpep) between the split luciferase fragments. The complementation bioluminescence of this reporter was dependent on the Src activity status. In our study, Src kinase activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to clinical small-molecular kinase inhibitors, dasatinib and saracatinib. This system was also applied for high-throughput screening of Src inhibitors against a kinase inhibitor library in living cells. These results provide unique insights into drug development and pharmacokinetics/phoarmocodynamics of therapeutic drugs targeting Src signaling pathway enabling the optimization of drug administration schedules for maximum benefit. Using both Firefly and Renilla luciferase imaging, we have successfully monitored Src tyrosine kinase activity and Akt serine/threonine kinase activity concurrently in one tumor xenograft. This dual luciferase reporter imaging system will be helpful in exploring the complex signaling networks in vivo. The strategies reported here can also be extended to study and image other important kinases and the cross-talks among them.
authors.affiliation: 1. Department of Medical Oncology, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China;; 2. Laboratory of Signal Transduction & Molecular Targeted Therapy, State Key Laboratory of Biotherapy, Sichuan University, Chengdu 610041, Sichuan, China;
authors.forename: Weibing
authors.initials: W
authors.lastname: Leng
authors.affiliation: 1. Department of Medical Oncology, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China;; 2. Laboratory of Signal Transduction & Molecular Targeted Therapy, State Key Laboratory of Biotherapy, Sichuan University, Chengdu 610041, Sichuan, China;
authors.forename: Dezhi
authors.initials: D
authors.lastname: Li
authors.affiliation: 1. Department of Medical Oncology, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China;
authors.forename: Liang
authors.initials: L
authors.lastname: Chen
authors.affiliation: 2. Laboratory of Signal Transduction & Molecular Targeted Therapy, State Key Laboratory of Biotherapy, Sichuan University, Chengdu 610041, Sichuan, China;
authors.forename: Hongwei
authors.initials: H
authors.lastname: Xia
authors.affiliation: 2. Laboratory of Signal Transduction & Molecular Targeted Therapy, State Key Laboratory of Biotherapy, Sichuan University, Chengdu 610041, Sichuan, China;
authors.forename: Qiulin
authors.initials: Q
authors.lastname: Tang
authors.affiliation: 1. Department of Medical Oncology, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China;
authors.forename: Baoqin
authors.initials: B
authors.lastname: Chen
authors.affiliation: 3. Department of Radiology, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China.
authors.forename: Qiyong
authors.initials: Q
authors.lastname: Gong
authors.affiliation: 3. Department of Radiology, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China.
authors.forename: Fabao
authors.initials: F
authors.lastname: Gao
authors.affiliation: 1. Department of Medical Oncology, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China;; 2. Laboratory of Signal Transduction & Molecular Targeted Therapy, State Key Laboratory of Biotherapy, Sichuan University, Chengdu 610041, Sichuan, China;
authors.forename: Feng
authors.initials: F
authors.lastname: Bi
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D017930:Genes, Reporter; D064593:Heterografts; D049410:Luciferases, Renilla / Q000032:analysis* / Q000235:genetics; D008163:Luminescent Measurements; D051379:Mice; D009369:Neoplasms / Q000000981:diagnostic imaging*; D017422:Sequence Analysis, DNA; D019061:src-Family Kinases / Q000032:analysis*
chemical_list: D049410:Luciferases, Renilla; D019061:src-Family Kinases
keywords: Src tyrosine kinase; bioluminescence imaging (BLI); drug development.; kinase activity; noninvasive molecular imaging