Analytical Sciences

Abstract − Analytical Sciences, 20(1), 61 (2004).

A New Luciferase Reporter Gene Assay for the Detection of Androgenic and Antiandrogenic Effects Based on a Human Prostate Specific Antigen Promoter and PC3/AR Human Prostate Cancer Cells
Ryoichi KIZU,*1,* Naoki OTSUKI,*3 Yoshiko KISHIDA,*1 Akira TORIBA,*3 Atsushi MIZOKAMI,*4 Kerry L. BURNSTEIN,*5 Carolyn M. KLINGE,*6 and Kazuichi HAYAKAWA*1,*2
*1 Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa 920-0934, Japan
*2 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Kawaguchi 332-0012, Japan
*3 Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa 920-0934, Japan
*4 Department of Urology, School of Medicine, Kanazawa University, Kanazawa 920-8641, Japan
*5 Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33101, USA
*6 Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, Kentucky 40292, USA
We developed a new mammalian cell-based luciferase reporter gene assay for androgenic and antiandrogenic activities of chemicals and environmental samples. Environmental samples usually have a complex matrix that may contain the constituents acting as androgen receptor (AR) agonists, AR antagonists or aryl hydrocarbon receptor (AhR) agonists. AhR agonists are known to elicit the antiandrogenic effect through cross-talk between AR and AhR signal transduction pathways. In this study, PC3/AR human prostate carcinoma cells were transiently transfected with a prostate-specific antigen (PSA) promoter-driven luciferase expression plasmid. The cells were treated with a test compound or an environmental sample for 24 h at 37°C and then measured for luciferase activity. The luciferase activity was induced by dihydrotestosterone (DHT) in a concentration-dependent manner in a concentration range from 10 fM to 1 nM. R1881, a synthetic androgen receptor agonist, induced luciferase activity and its inductive effects was additive to that of DHT. The luciferase activity was not induced by cortisol, a glucocorticoid, progesterone, a progestin, and 17 β-estradiol, an estrogen in a concentration range of up to 1 μM. DHT-induced luciferase activity was reduced by bicalutamide and cyproterone acetate, AR antagonists, and also by benzo[a]pyrene, an aryl hydrocarbon receptor agonist, through AhR-mediated pathways. All of these findings indicate that the present assay system correctly responds to AR agonists, AR antagonists and AhR agonist and, therefore, it is a powerful tool for the sensitive and selective screening of chemicals and environmental samples for their androgenic and antiandrogenic activities. We developed the first assay system, in which the expression of luciferase was driven by the promoter of a prostate-specific antigen gene, a typical human androgenregulated gene.