Year-end Sale % OFF 
Abstract
Activation of beta-catenin/T cell factor (TCF) transcription as a result of mutations in the adenomatous polyposis coli (APC) and/or beta-catenin genes occurs in the majority of colon tumors. An increasing number of genes, including c-myc and cyclin D1, have been implicated as targets of this pathway. We now report that the dominant negative helix-loop-helix regulator Id2 is also a target of the beta-catenin/TCF transcription pathway in colon adenocarcinoma. Investigation of the mechanism for the overexpression of Id2 in colon carcinoma cells demonstrated that the Id2 promoter is activated, and the Id2 protein is up-regulated by beta-catenin. Conversely, reducing free beta-catenin blocked this induction of promoter activity. We have also used an electrophoretic mobility shift assay and supershift to identify a motif in the Id2 promoter that binds to TCF4 protein. Site-directed mutagenesis of this motif abolished promoter reporter activity. Both transfection of Id2 into SW480 cells and induction of Id2 in HT29 colon cells was found to increase anchorage-independent survival of these cells. Growing evidence associates disruption to Id2 expression with tumorigenesis, and our findings suggest that this dysregulation of Id2 expression is due to the activation of the beta-catenin/TCF pathway.
Highlights
Mutations in the adenomatous polyposis coli (APC) and -catenin genes are common in human colonic cancer [1, 2]
We have demonstrated that Id2 is a target of the -catenin/T cell factor (TCF) pathway
The pattern of Id2 expression correlates with the pattern of -catenin expression
Summary
Tissue Samples—Samples of tumor, polyp, and normal tissue were obtained from patients undergoing Surgery at Western Hospital, Victoria, Australia. Membranes were blocked with 5% (w/v) nonfat dry milk in Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBS-T) and incubated with anti-Id2 rabbit polyclonal antiserum Gel Retardation Assays—Id2 promoter binding of in vitro synthesized protein (TNT, Promega Corp.) or nuclear lysates were assessed by electrophoretic mobility shift assay following the methods described previously [14]. HT29 empty vector and Id2 sense and antisense cell lines were plated in semisolid agar as described above and cultured with and without induction by the addition of 2 g/ml doxycycline
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
