Induction of cre recombinase activity using modified androgen receptor ligand binding domains: a sensitive assay for ligand-receptor interactions.
Novel systems of inducible gene expression are presented in which CRE-M, an altered form of cre recombinase (cre), is fused to and activated by ligand binding to two forms of the androgen receptor (AR) ligand binding domain (LBD). Selective activation or inactivation of gene transcription is induced upon the addition of appropriate ligand. The coupling of this cre-LBD system with our previously reported highly sensitive assay to measure cre activity in vitro using a dual fluorescent gene switch reporter provides a novel, high-throughput assay system for identifying compounds that bind to and activate various forms of the LBD of androgen receptor. This method can similarly be applied to screen compounds for their activating properties on other steroid hormone LBDs. Three different forms of the AR-LBD were fused to CRE-M, including the wild-type AR-LBD (wt), a non-ligand binding truncated form, LBD (T), and a mutated form (Thr-->Ala substitution) identified in the LNCaP prostate cancer cell line, LBD (LNCaP). We demonstrate a 10-fold induction of cre activity by the addition of androgen agonists to the CRE-M-AR-LBD(wt) fusion protein, but not in the presence of the anti-androgen, flutamide. However, cre activity can be induced by flutamide with the CRE-M-AR-LBD(LNCaP) fusion protein. Similar activation properties were obtained when these fusion proteins were expressed using adenoviral vectors. When combined with our previously reported cre-lox gene switch system, the CRE-M-AR-LBD system can be utilized in gene therapy systems in which a therapeutic product may be initially expressed, replaced by a second product, or turned-off following exposure to ligand. This provides an important, additional level of regulation to gene therapy systems.
Kaczmarczyk
Stanislaw J
SJ
Transgenic Oncogenesis Group, Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Bethesda, MD 20892, USA.
Green
Jeffrey E
JE
D000256:Adenoviridae / Q000235:genetics; D000726:Androgen Antagonists / Q000494:pharmacology; D000728:Androgens / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D005821:Genetic Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D019426:Integrases / Q000235:genetics* / Q000378:metabolism*; D008024:Ligands; D008856:Microscopy, Fluorescence; D017434:Protein Structure, Tertiary; D011944:Receptors, Androgen / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015533:Transcriptional Activation; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*
D000726:Androgen Antagonists; D000728:Androgens; D008024:Ligands; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases