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Sensitive in vitro test systems to determine androgenic/antiandrogenic activity
Author(s) -
Guth Sabine E.,
Böhm Sonja,
Mußler Bernd H.,
Eisenbrand Gerhard
Publication year - 2004
Publication title -
molecular nutrition and food research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.495
H-Index - 131
eISSN - 1613-4133
pISSN - 1613-4125
DOI - 10.1002/mnfr.200400023
Subject(s) - transactivation , dihydrotestosterone , reporter gene , androgen receptor , luciferase , microbiology and biotechnology , androgen , biology , chemistry , endocrinology , transfection , medicine , gene expression , prostate cancer , hormone , gene , biochemistry , cancer
We report on the establishment of one transgenic and two endogenous reporter gene assays to determine androgenic/antiandrogenic activity. A transient transactivation assay was developed in COS‐7 African green monkey kidney cells. Three plasmids were co‐transfected by electroporation: the human androgen receptor expression vector pSG5AR, the reporter gene vector pMamneoLuc, expressing luciferase under the control of the mouse mammary tumor virus (MMTV) promotor containing 4 hormone responsive elements (HREs), and the control plasmid pSVβ . Transcriptional activation was measured by luciferase‐mediated chemoluminescence. In T47D human breast cancer cells two endogenous reporter gene systems were established: one based on the androgen‐induced expression of prostate‐specific antigen (PSA), the other based on the androgen‐repressed expression of testosterone repressed message 2 (TRPM‐2). PSA protein was measured by enzyme‐linked immunosorbent assay (ELISA), TRPM‐2 m‐RNA by reverse transcriptase polymerase chain reaction (RT‐PCR). All three test systems were validated using the physiological androgen dihydrotestosterone (DHT) as agonist and the established antiandrogens hydroxyflutamide and p,p′‐dichlorodiphenylethene (p,p′‐DDE) as antagonists. The PSA assay was the most sensitive test system with an EC 50 of 0.7 n M for DHT‐induced response. The transient transactivation assay in COS‐7 cells was less sensitive with an EC 50 of 9.7 n M DHT. In the PSA assay hydroxyflutamide was a more potent antagonist (IC 30 = 0.02 μ M ) than p,p′‐DDE (IC 30 = 0.9 μ M ). Similar results were obtained in the TRPM‐2 assay. In the transient transactivation assay in COS‐7 cells, both compounds elicited about 30% of the agonistic response induced by 100 nM DHT, thus showing partial agonistic properties. In summary, three highly sensitive and complementary in vitro test systems, together achieving enhanced specificity for detection and assessment of androgenic/antiandrogenic activity have been established and validated.