Fluorescence-based functional assay for Wnt/beta-catenin signaling activity.
Aberrant activation of beta-catenin signaling has been implicated in the development of human cancers. As a Wnt signal transducer, beta-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 beta-catenin-mediated heterologous transactivation system, which consisted of a chimeric transcription factor constructed by fusing the GAL4 DNA-binding domain with the full-length beta-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 beta-catenin in a Wnt-responsive fashion. Thus, this transactivation system could be used as a functional reporter to identify potential upstream factors that deregulate beta-catenin signaling during tumorigenesis, as well as to screen for potential anti-cancer agents that specifically inhibit beta-catenin signaling in human tumors.
Zhou
L
L
The University of Chicago Medical Center, IL, USA.
An
N
N
Jiang
W
W
Haydon
R
R
Cheng
H
H
Zhou
Q
Q
Breyer
B
B
Feng
T
T
He
T-C
TC
D000230:Adenocarcinoma / Q000473:pathology; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001859:Bone Neoplasms; D002460:Cell Line; D002813:Chondrosarcoma / Q000473:pathology; D003110:Colonic Neoplasms / Q000473:pathology; D017077:Culture Media, Conditioned / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D004268:DNA-Binding Proteins; D005470:Fluorometry / Q000379:methods*; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D007668:Kidney; D008156:Luciferases / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D008164:Luminescent Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000502:physiology; D012516:Osteosarcoma / Q000473:pathology; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D015398:Signal Transduction; D015534:Trans-Activators / Q000037:antagonists & inhibitors / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured; D051153:Wnt Proteins; D051155:Wnt1 Protein; D029961:Zebrafish Proteins; D051176:beta Catenin
C495270:CTNNB1 protein, human; C495267:CTNNB1 protein, mouse; D017077:Culture Media, Conditioned; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D008164:Luminescent Proteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C495247:WNT1 protein, human; D051153:Wnt Proteins; D051155:Wnt1 Protein; C495246:Wnt1 protein, mouse; D029961:Zebrafish Proteins; D051176:beta Catenin; D049452:Green Fluorescent Proteins; D008156:Luciferases