Abstract 724: New fluorescence-based cell assays for visualizing the Raf - Ras interaction and the Wnt pathway activity.

Abstract

Abstract Colorectal cancer (CRC) belongs to the most frequent cancer types (Hollande et al. 2010). As molecular mechanism for CRC, several mechanisms are presumed, including chromosome instability going along with the inactivation of the adenomatous polyposis coli (APC) gene, a key protein of Wnt signaling (Thorstensen et al. 2005). It is widely accepted that an abnormally activated Wnt pathway is a major cause for non-braked proliferation and for reduced cell differentiation. The second major cause of CRC is the deregulation of genes affecting the Ras pathway (Jeremy R. Jass, 2006). Nevertheless, specific drugs targeting either the Wnt or the Ras pathways are rare. Therefore, we designed new, fluorescence-based reporter gene assays either for the Wnt or the Ras/Raf pathway as tools to simplify the screening for compounds modifying each pathway. Such cellular systems could be applicable for high-throughput-screening as well as Live-Cell-Imaging of compound effects, as shown in preliminary experiments. Therein, stably transfected HEK 293T cells bearing the new, Wnt-responsive reporter gene vector, were treated with LiCl, Wnt3a protein or acetylsalicylic acid in order to get a specific response. Cells showed an increase of the fluorescence level, which is measurable by a standard ELISA reader and could be visualized via fluorescence microscopy. In MDCK or MDCK-F3 cells bearing the Raf-responsive element, treated with EGF or a Raf inhibitor, the localization of the fluorescence signal changed. Moreover, treating the cells with different combination of the mentioned Wnt- or Ras-active compounds also showed a response, indicating a correlation between the two pathways. These results lead to the conclusion to combine both reporter gene vectors in order to detect the interplay of the pathways. Besides sensibilization of the assays and setting up the Live-Cell imaging experiments, this will be our further steps. In summary, we designed several assays applicable in a screening assay for new, specific compounds targeting the two most important cancer-related pathways Wnt and Ras/Raf. Such an assay is more cost-effective than conventional assays, and also offers the possibility to characterize directly the biochemical mechanisms and interplays via Live-Cell-Imaging. Citation Format: Johanna N. Apfel, Patricia T. Reischmann. New fluorescence-based cell assays for visualizing the Raf - Ras interaction and the Wnt pathway activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 724. doi:10.1158/1538-7445.AM2013-724