A protein folding molecular imaging biosensor monitors the effects of drugs that restore mutant p53 structure and its downstream function in glioblastoma cells
Year: 2018
Type: article
Abstract: // Ramasamy Paulmurugan 1 , Rayhaneh Afjei 2 , Thillai V. Sekar 1 , Husam A. Babikir 2 and Tarik F. Massoud 2 1 Cellular Pathway Imaging Laboratory (CPIL), Molecular Imaging Program at Stanford, Stanford University School of Medicine, Palo Alto, CA 94305, USA 2 Laboratory for Experimental and Molecular Neuroimaging (LEMNI), Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, CA 94305, USA Correspondence to: Tarik F. Massoud, email: [email protected] Ramasamy Paulmurugan, email: [email protected] Keywords: drug screening; luciferase; misfolding; Renilla; temozolomide Received: January 16, 2018 Accepted: March 15, 2018 Published: April 20, 2018 ABSTRACT Misfolding mutations in the DNA-binding domain of p53 alter its conformation, affecting the efficiency with which it binds to chromatin to regulate target gene expression and cell cycle checkpoint functions in many cancers, including glioblastoma. Small molecule drugs that recover misfolded p53 structure and function may improve chemotherapy by activating p53-mediated senescence. We constructed and optimized a split Renilla luciferase (RLUC) complementation molecular biosensor (NRLUC-p53-CRLUC) to determine small molecule-meditated folding changes in p53 protein. After initial evaluation of the biosensor in three different cells lines, we engineered endogenously p53 P98L mutant (i.e. not affecting the DNA-binding domain) Ln229 glioblastoma cells, to express the biosensor containing one of four different p53 proteins: p53 wt , p53 Y220C , p53 G245S and p53 R282W . We evaluated the consequent phenotypic changes in these four variant cells as well as the parental cells after exposure to PhiKan083 and SCH529074, drugs previously reported to activate mutant p53 folding. Specifically, we measured induced RLUC complementation and consequent therapeutic response. Upon stable transduction with the p53 biosensors, we demonstrated that these originally p53 P98L Ln229 cells had acquired p53 cellular phenotypes representative of each p53 protein expressed within the biosensor fusion protein. In these engineered variants we found a differential drug response when treated with doxorubicin and temozolomide, either independently or in combination with PhiKan083 or SCH529074. We thus developed a molecular imaging complementation biosensor that mimics endogenous p53 function for use in future applications to screen novel or repurposed drugs that counter the effects of misfolding mutations responsible for oncogenic structural changes in p53.
Source: Oncotarget
Institutions: Stanford Medicine, Stanford University
Language: English
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: diamond
Funder: Ben and Catherine Ivy Foundation
OpenAlex ID: W2802536440