Abstract 5228: Bioluminescent reporter assays to identify signaling pathways in vivo

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Whole animal optical imaging provides new ways to detect tumor cells and other biological activities in the system. Bioluminescent imaginghas advantages over fluorescent imaging since animal tissues have less autoluminescence. Due to high signal to background ratio, bioluminescent imagingcan detect subtle changes of light emission in the animals. Luciferasesare the most popular enzymes for optical imaging. Lights are produced by enzymatic reactions in the presence of substrates. Commonly used luciferaseswere cloned from firefly ( Photinus pyralis) and Sea Pansy(Renilla reniformis). Firefly and Renilla luciferaseuses D- luciferinand coelenterazineas a substrate, respectively. Since these luciferasesuse different kinds of substrates, they can be used in a reporter assay simultaneously. Bioluminescentreporter assays can be achieved by conjugating transcription factor responding elements and a luciferasereporter gene. Upon signals from the outside of the cells, luciferaseexpression 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 lentivirusreporters that carry destabilized firefly luciferasealong 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 luciferasereporter 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][1] or PD98059 was used for different duration. Bioluminescent imageswere taken at multiple time points and light emissions were quantitated. Among the compound tested, LPS generated more than 50 fold increase of bioluminescencein NF-kB luciferase(NFkB-luc) reporter cell line in vitro. In addition, the assay showed dose-dependent response. Renilla luciferaseactivities 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 imageswere taken using a cooled CCD camera. Our results showed that animals implanted with NFkB-luc mice showed 6 fold increase of luciferaseactivity while TGFb-luc or Neg-luc implanted animals showed little change in luciferaseactivity. Present study demonstrates that bioluminescentreporter 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. [1]: /lookup/external-ref?link_type=GEN&access_num=SB203580&atom=%2Fcanres%2F70%2F8_Supplement%2F5228.atom