title: Molecular engineering of a two-step transcription amplification (TSTA) system for transgene delivery in prostate cancer.
abstract: Gene therapy is founded on the concept that tissue-specific promoters can express heterologous genes for molecular imaging or therapeutic applications. The engineering of cell-specific enhancers to improve potency and the development of two-step transcriptional activation (TSTA) approaches have significantly improved the efficacy of transgene expression. Here we combine these technologies to create a robust, titratable, androgen-responsive system targeted to prostate cancer cells. Our "chimeric" TSTA system uses a duplicated variant of the prostate-specific antigen (PSA) gene enhancer to express GAL4 derivatives fused to one, two, or four VP16 activation domains. We targeted the resulting activators to cells with reporter templates bearing one, two, or five GAL4 binding sites upstream of firefly luciferase. We monitored activity via firefly luciferase assays in transfected cell extracts and in live nude mice using a cooled charge-coupled device (CCD) imaging system. In this system, we found that firefly luciferase expression in prostate cancer cells can be varied over an 800-fold range. We also found that a single plasmid bearing the optimized enhancer, GAL4-VP16 derivative, and reporter expressed firefly luciferase at 20-fold higher levels than the cytomegalovirus enhancer. We discuss the implications of this strategy and its application to molecular imaging and therapy.
authors.affiliation: Department of Biological Chemistry, University of California, Los Angeles School of Medicine, Los Angeles, California 90095-1737, USA.
authors.forename: Liqun
authors.initials: L
authors.lastname: Zhang
authors.forename: Jason Yeates
authors.initials: JY
authors.lastname: Adams
authors.forename: Erika
authors.initials: E
authors.lastname: Billick
authors.forename: Romyla
authors.initials: R
authors.lastname: Ilagan
authors.forename: Meera
authors.initials: M
authors.lastname: Iyer
authors.forename: Kim
authors.initials: K
authors.lastname: Le
authors.forename: Andrea
authors.initials: A
authors.lastname: Smallwood
authors.forename: Sanjiv S
authors.initials: SS
authors.lastname: Gambhir
authors.forename: Michael
authors.initials: M
authors.lastname: Carey
authors.forename: Lily
authors.initials: L
authors.lastname: Wu
mesh_terms: D000818:Animals; D004742:Enhancer Elements, Genetic; D005784:Gene Amplification; D018014:Gene Transfer Techniques; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D008297:Male; D051379:Mice; D008819:Mice, Nude; D021141:Nucleic Acid Amplification Techniques; D011401:Promoter Regions, Genetic; D011471:Prostatic Neoplasms / Q000235:genetics / Q000628:therapy*; D017434:Protein Structure, Tertiary; D014018:Tissue Distribution; D014158:Transcription, Genetic; D019076:Transgenes