Fluorescence-Based Functional Assay for Wnt/β-Catenin Signaling Activity

• Published in: BioTechniques Vol. 33; no. 5; pp. 1126 - 1135
• Main Authors: Zhou, L, An, N, Jiang, W, Haydon, R, Cheng, H, Zhou, Q, Breyer, B, Feng, T, He, T.-C
• Format: Journal Article
• Language: English
• Published: England Future Science Ltd 01.11.2002; Taylor & Francis Group
• Subjects: Adenocarcinoma - pathology; Adenoviridae - genetics; Animals; beta Catenin; Bone Neoplasms; Cell Line; Chondrosarcoma - pathology; Colonic Neoplasms - pathology; Culture Media, Conditioned - pharmacology; Cytoskeletal Proteins - antagonists & inhibitors; Cytoskeletal Proteins - physiology; DNA-Binding Proteins; Fluorometry - methods; Genes, Reporter; Genetic Vectors - genetics; Green Fluorescent Proteins; Humans; Kidney; Luciferases - analysis; Luciferases - biosynthesis; Luciferases - genetics; Luminescent Proteins - analysis; Luminescent Proteins - biosynthesis; Luminescent Proteins - genetics; Mice; Neoplasm Proteins - antagonists & inhibitors; Neoplasm Proteins - physiology; Osteosarcoma - pathology; Protein Structure, Tertiary; Proto-Oncogene Proteins - physiology; Recombinant Fusion Proteins - physiology; Saccharomyces cerevisiae Proteins - genetics; Signal Transduction; Trans-Activators - antagonists & inhibitors; Trans-Activators - physiology; Transcription Factors - genetics; Transcriptional Activation; Transfection; Tumor Cells, Cultured; Wnt Proteins; Wnt1 Protein; Zebrafish Proteins
• ISSN: 0736-6205; 1940-9818
• DOI: 10.2144/02335dd07

Aberrant activation of β-catenin signaling has been implicated in the development of human cancers. As a Wnt signal transducer, β-catenin forms a complex with the lymphocyte enhancer-binding factor/T cell factor transcription factor and activates downstream targets that promote cell proliferation. Here we developed a Wnt-dependent β-catenin-mediated heterologous transactivation system, which consisted of a chimeric transcription factor constructed by fusing the GAL4 DNA-binding domain with the full-length β-catenin, and a GAL4-responsive reporter expressing GFP. The chimeric transcription factor was highly unstable and exerted no detectable transactivating effect on the GAL4-responsive reporter. However, lithium and Wnt1 significantly stabilized this chimeric transactivator, indicating that this transactivation system is regulated by β-catenin in a Wnt-responsive fashion. Thus, this transactivation system could be used as a functional reporter to identify potential upstream factors that deregulate β-catenin signaling during tumorigenesis, as well as to screen for potential anti-cancer agents that specifically inhibit β-catenin signaling in human tumors.