A Novel Reporter System for Molecular Imaging and High-Throughput Screening of Anticancer Drugs

Authors

  • Dr. Jingping Xie,

    Corresponding author
    1. Institute of Imaging Science, Vanderbilt University, Nashville, TN 37232 (USA)
    • Institute of Imaging Science, Vanderbilt University, Nashville, TN 37232 (USA)
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    • These authors contributed equally to this work.

  • Dr. Chunxia Wang,

    1. Institute of Imaging Science, Vanderbilt University, Nashville, TN 37232 (USA)
    2. Current address: Department of Pharmacy, Nanfang Hospital, Southern Medical University, 1838 North Guangzhou Avenue, Guangzhou, Guangdong 510515 (China)
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    • These authors contributed equally to this work.

  • Dr. John Virostko,

    1. Institute of Imaging Science, Vanderbilt University, Nashville, TN 37232 (USA)
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  • Dr. H. Charles Manning,

    1. Institute of Imaging Science, Vanderbilt University, Nashville, TN 37232 (USA)
    2. Department of Radiology and Radiological Sciences, Vanderbilt University, Nashville, TN 37232 (USA)
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  • Dr. Wellington Pham,

    1. Institute of Imaging Science, Vanderbilt University, Nashville, TN 37232 (USA)
    2. Department of Radiology and Radiological Sciences, Vanderbilt University, Nashville, TN 37232 (USA)
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  • Dr. Joshua Bauer,

    1. HTS Core lab, Institute for Chemical Biology, Vanderbilt University, Nashville, TN 37232 (USA)
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  • Prof. Dr. John C. Gore

    Corresponding author
    1. Institute of Imaging Science, Vanderbilt University, Nashville, TN 37232 (USA)
    2. Department of Radiology and Radiological Sciences, Vanderbilt University, Nashville, TN 37232 (USA)
    3. Department of Biomedical Engineering, Department of Physics and Astronomy, Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232 (USA)
    • Institute of Imaging Science, Vanderbilt University, Nashville, TN 37232 (USA)
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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.

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