Abstract 5228: Bioluminescent reporter assays to identify signaling pathways in vivo
Authors: Eva Barton, Angel Ang, Konnie Urban, Kevin Francis, Rajendra Singh, Mark Roskey, Dave Martz, Jae-Beom Kim
Journal: Cancer Research (2010)

Abstract Whole animal optical imaging provides new ways to detect tumor cells and other biological activities in the system. Bioluminescent imaging has advantages over fluorescent imaging since animal tissues have less autoluminescence. Due to high signal to background ratio, bioluminescent imaging can detect subtle changes of light emission in the animals. Luciferases are the most popular enzymes for optical imaging. Lights are produced by enzymatic reactions in the presence of substrates. Commonly used luciferases were cloned from firefly (Photinus pyralis) and Sea Pansy (Renilla reniformis). Firefly and Renilla luciferase uses D-luciferin and coelenterazine as a substrate, respectively. Since these luciferases use different kinds of substrates, they can be used in a reporter assay simultaneously. Bioluminescent reporter assays can be achieved by conjugating transcription factor responding elements and a luciferase reporter gene. Upon signals from the outside of the cells, luciferase expression can be regulated. Using multiple responding elements for unique transcription factors, one can identify signaling pathways that are responsive to drug treatments. We have tested 10 different lentivirus reporters that carry destabilized firefly luciferase along with basal promoter element joined to tandem repeats of distinct transcription responding element. PC3M human prostate cancer cells were permanently transfected and stable cell lines were generated. As a control, constitutively active Renilla luciferase reporter was co-transfected to each reporter line. In order to identify signaling pathways that are responding to drug treatment, we plated equal number of reporter cells in well plates and treated cells with various compounds. Cisplatin, lipopolysaccaride (LPS), Paclitaxel, PMA, SB203580 or PD98059 was used for different duration. Bioluminescent images were taken at multiple time points and light emissions were quantitated. Among the compound tested, LPS generated more than 50 fold increase of bioluminescence in NF-kB luciferase (NFkB-luc) reporter cell line in vitro. In addition, the assay showed dose-dependent response. Renilla luciferase activities remained same in all reporter cell lines. To validate in vivo response, PC3M NFkB-luc cells were implanted into male nu/nu mice subcutaneously. As controls, we also implanted a non-responding reporter upon LPS treatment (TGFb-luc) and negative control without responding elements (Neg-luc). Animals were administered with LPS and in vivo bioluminescence images were taken using a cooled CCD camera. Our results showed that animals implanted with NFkB-luc mice showed 6 fold increase of luciferase activity while TGFb-luc or Neg-luc implanted animals showed little change in luciferase activity. Present study demonstrates that bioluminescent reporter assays can be applied to elucidate the mode of action of a drug. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5228.