title: Molecular Imaging Biosensor Monitors p53 Sumoylation in Cells and Living Mice.
abstract: Small molecule mediated stabilization of p53 tumor suppressor protein through sumoylation is a promising new strategy for improving cancer chemotherapy. A molecular tool that monitors p53 sumoylation status and expedites screening for drugs that enhance p53 sumoylation would be beneficial. We report a molecularly engineered reporter fragment complementation biosensor based on optical imaging of Firefly luciferase (FLuc), to quantitatively image p53 sumoylation and desumoylation in cells and living mice. We initially characterized this biosensor by successfully imaging sumoylation of several target proteins, achieving significant FLuc complementation for ERα (p < 0.01), p53 (p < 0.005), FKBP12 (p < 0.03), ID (p < 0.03), and HDAC1 (p < 0.002). We then rigorously tested the sensitivity and specificity of the biosensor using several variants of p53 and SUMO1, including deletion mutants, and those with modified sequences containing the SUMO-acceptor site of target proteins. Next we evaluated the performance of the biosensor in HepG2 cells by treatment with ginkgolic acid, a drug that reduces p53 sumoylation, as well as trichostatin A, a potential inducer of p53 sumoylation by enhancement of its nuclear export. Lastly, we demonstrated the in vivo utility of this biosensor in monitoring and quantifying the effects of these drugs on p53 sumoylation in living mice using bioluminescence imaging. Adoption of this biosensor in future high throughput drug screening has the important potential to help identify new and repurposed small molecules that alter p53 sumoylation, and to preclinically evaluate candidate anticancer drugs in living animals.
authors.affiliation: Molecular Imaging Program at Stanford, Bio-X Program, Stanford University School of Medicine , Palo Alto, California 94304, United States.
authors.forename: Thillai V
authors.initials: TV
authors.lastname: Sekar
authors.affiliation: Molecular Imaging Program at Stanford, Bio-X Program, Stanford University School of Medicine , Palo Alto, California 94304, United States.
authors.forename: Kira
authors.initials: K
authors.lastname: Foygel
authors.affiliation: Molecular Imaging Program at Stanford, Bio-X Program, Stanford University School of Medicine , Palo Alto, California 94304, United States.
authors.forename: Rammohan
authors.initials: R
authors.lastname: Devulapally
authors.affiliation: Radiation Oncology, Stanford University School of Medicine , Stanford, California 94305, United States.
authors.forename: Vineet
authors.initials: V
authors.lastname: Kumar
authors.affiliation: Radiation Oncology, Stanford University School of Medicine , Stanford, California 94305, United States.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Malhotra
authors.affiliation: Molecular Imaging Program at Stanford, Bio-X Program, Stanford University School of Medicine , Palo Alto, California 94304, United States.
authors.forename: Tarik F
authors.initials: TF
authors.lastname: Massoud
authors.affiliation: Molecular Imaging Program at Stanford, Bio-X Program, Stanford University School of Medicine , Palo Alto, California 94304, United States.
authors.forename: Ramasamy
authors.initials: R
authors.lastname: Paulmurugan
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation; D056945:Hep G2 Cells; D006801:Humans; D049409:Luciferases, Firefly / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D008958:Models, Molecular; D057054:Molecular Imaging; D058207:Sumoylation; D016159:Tumor Suppressor Protein p53 / Q000032:analysis* / Q000378:metabolism
chemical_list: D016159:Tumor Suppressor Protein p53; D049409:Luciferases, Firefly