title: A novel reporter system for molecular imaging and high-throughput screening of anticancer drugs.
abstract: Apoptosis is irreversible programmed cell death, characterized by a cellular cascade activation of caspase 3, which subsequently degrades proteins and other components of cells with a motif sequence. Here we report a novel reporter system to detect apoptosis, growth arrest, and cell death based on controlled and self-amplified protein degradation. The key element of the reporter system is an apoptotic sensor chimerical protein which consists of three components: procaspase 3, ubiquitin (Ub), and a strong consensus sequence of N-degron. Between each of these units is a DEVD (Asp-Glu-Val-Asp) sequence, which acts as the cleavage target of caspase 3. This non-conventional signal loss approach is much more sensitive than other native methods that are based on signal gain. The superior sensitivity is demonstrated by its effective application in 386-well high-throughput screening (HTS) with low drug concentrations and a short incubation time. The HTS selection process using this reporter system is very simple and economic. The simplicity eliminates potential errors introduced by multiple steps; there is no need for any substrate. Furthermore, the cells in the assay need not be disrupted, and the morphology of the cells can provide additional information on mechanisms. After HTS, the intact cells can also be used for other analytic analysis. This system thus has a potentially important role in the discovery and development of new anticancer drugs. It also appears to be very versatile, can be used both in vitro and in vivo with different linked reporter genes, and can be used for a variety of imaging applications.
authors.affiliation: Institute of Imaging Science, Vanderbilt University, Nashville, TN 37232, USA. Jingping.Xie@vanderbilt.edu
authors.forename: Jingping
authors.initials: J
authors.lastname: Xie
authors.forename: Chunxia
authors.initials: C
authors.lastname: Wang
authors.forename: John
authors.initials: J
authors.lastname: Virostko
authors.forename: H Charles
authors.initials: HC
authors.lastname: Manning
authors.forename: Wellington
authors.initials: W
authors.lastname: Pham
authors.forename: Joshua
authors.initials: J
authors.lastname: Bauer
authors.forename: John C
authors.initials: JC
authors.lastname: Gore
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D001681:Biological Assay; D002460:Cell Line; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D055808:Drug Discovery; D004354:Drug Screening Assays, Antitumor; D017930:Genes, Reporter / Q000187:drug effects*; D006801:Humans; D018613:Microscopy, Confocal; D011994:Recombinant Proteins / Q000235:genetics; D015203:Reproducibility of Results
chemical_list: D000970:Antineoplastic Agents; D011994:Recombinant Proteins
keywords: apoptosis; caspase; drug discovery; high-throughput screening; molecular imaging