Journal of Steroid Biochemistry & Molecular Biology 101 (2006) 68–77 Determination of androgen bioactivity in human serum samples using a recombinant cell based in vitro bioassay Partha Roy 1 , Stephen Franks, Moira Read, Ilpo T. Huhtaniemi ∗ Institute of Reproductive and Developmental Biology, Faculty of Medicine, Imperial College London, Du Cane Road, London W12 ONN, UK Abstract The present study describes the development and optimization of a cell-based reporter assay for the determination of androgen bioactivity levels in human serum samples. Towards this end, human embryonic kidney (HEK) 293 cells were cotransfected with two plasmids, one encoding the mouse mammary tumor virus (MMTV)-driven luciferase reporter gene and the other the SV40 promoter-driven human androgen receptor (AR), and a stable cell line, expressing human AR and androgen-responsive luciferase was established by antibiotic selection. RT-PCR confirmed proper transcription and stable integration of AR in the cell line. On stimulation of the cells with testosterone (T) for 24 h, luciferase activity was increased in dose-dependent fashion up to 15-fold, with the minimum effective concentration of T being 0.03nmol/l. T-induced reporter gene expression of the cells was inhibited by the anti-androgens cyproterone acetate and hydroxyflutamide. Upon stimulation with non-androgenic steroids like estradiol, progesterone, dexamethasone and cortisol, the cells showed only marginal activity except for a weak glucocorticoid effect at high concentration. The cells also responded well to various chemicals (mostly pesticides, their metabolites and common industrial chemicals) with known androgenic activity. The cell line was further optimized by measuring the levels of androgens from male and female serum samples. Our data indicated that the cells can be used to estimate androgen bioactivity in serum of females suffering from polycystic ovary syndrome (PCOS) and males of different age groups. In conclusion, we demonstrate that the bioassay based on this cell line provides a reliable method to determine serum levels of androgen bioactivity from biological samples even at the low concentrations present in female circulation. The 96-well plate format makes the assay suitable for high throughput measurements. © 2006 Elsevier Ltd. All rights reserved. Keywords: Bioassay; Androgen; Androgen receptor; Polycystic ovarian syndrome; Luciferase 1. Introduction Steroid hormones, ligands for the ligand-dependent tran- scription factors, mediate some of the most important phys- iological functions of the body. Upon ligand binding, the steroid hormone receptors interact directly with DNA, thus regulating the activity of their target genes. Androgens, a sub- group of steroid hormones, play a key role in the development and maintenance of male sex characteristics. Their biologi- cal effects are mediated by the androgen receptor (AR), one of the ligand-dependent transcription factors, which is ubiq- uitously expressed in the body. The levels of AR vary in ∗ Corresponding author. Tel.: +44 20 7594 2104; fax: +44 20 7594 2184. E-mail address: ilpo.huhtaniemi@imperial.ac.uk (I.T. Huhtaniemi). 1 Present address: Department of Biotechnology, Indian Institute of Tech- nology Roorkee, Roorkee 247 667, Uttaranchal, India. different physiological and pathological conditions such as malignancies or in response to physiological changes of the endocrine system. Upon entering the target cell androgens bind to AR and the cytosolic hormone-receptor complex is translocated to the nucleus where it binds to the regulatory regions of androgen-responsive genes as a homodimer and subsequently stimulates their transcription [1,2]. AR belongs to the nuclear receptor superfamily consist- ing of receptors for Vitamin D 3 , thyroid hormones, retinoids and steroid hormones [3,4]. These receptors have conserved DNA- and ligand binding domains (DBD and LBD, respec- tively) and variable hinge and N-terminal regions [3]. For AR, the N-terminal region encompasses the primary transcrip- tional activation domain. Upon androgen binding, the LBD and the N-terminal region of AR have been shown to interact, which is known to activate the process of AR dimerization [4–6], modulate transcriptional activity [7] and stabilize the receptor at low ligand concentrations [8]. 0960-0760/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2006.06.014