A novel estrogen receptor intramolecular folding-based titratable transgene expression system.
The use of regulated gene expression systems is important for successful gene therapy applications. In this study, ligand-induced structural change in the estrogen receptor (ER) was used to develop a novel ER intramolecular folding-based transcriptional activation system. The system was studied using ER-variants of different lengths, flanked on either side by the GAL4-DNA-binding domain and the VP16-transactivation domain (GAL4(DBD)-ER-VP16). The ER ligands of different types showed efficient ligand-regulated transactivation. We also characterized a bidirectional transactivation system based on the ER and demonstrated its utility in titrating both reporter and therapeutic gene expression. The ligand-regulated transactivation system developed by using a mutant form of the ER (G521T, lacking affinity for the endogenous ligand 17beta-estradiol, whereas maintaining affinity for other ligands) showed efficient activation by the ligand raloxifene in living mice without significant interference from the circulating endogenous ligand. The ligand-regulated transactivation system was used to test the therapeutic efficiency of the tumor suppressor protein p53 in HepG2 (p53(+/+)) and SKBr3 (p53(-/-)/mutant-p53(+/+)) cells in culture and tumor xenografts in living mice. The multifunctional capabilities of this system should be useful for gene therapy applications, to study ER biology, to evaluate gene regulation, ER ligand screening, and ER ligand biocharacterization in cells and living animals.
Paulmurugan
Ramasamy
R
Department of Radiology, Stanford University School of Medicine, James H. Clark Center, 318 Campus Drive, 150 East Wing, 1st Floor, Stanford, CA 94305-5427, USA. paulmur8@stanford.edu
Padmanabhan
Parasuraman
P
Ahn
Byeong-Cheol
BC
Ray
Sunetra
S
Willmann
Juergen K
JK
Massoud
Tarik F
TF
Biswal
Sandip
S
Gambhir
Sanjiv S
SS
D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D005260:Female; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D006801:Humans; D051379:Mice; D011485:Protein Binding; D017510:Protein Folding; D011960:Receptors, Estrogen / Q000737:chemistry* / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics*; D019076:Transgenes / Q000235:genetics*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D011960:Receptors, Estrogen; D016159:Tumor Suppressor Protein p53