Cell-Based Assays for Screening Androgen Receptor Ligands

Abstract

The androgen receptor (AR, NR3C4) mediates the majority of androgen effects on target cells. The AR is activated following ligand binding that result is enhanced of target gene transcription. Several cell-based model systems have been developed that allow sensitive detection and monitoring of steroids or other compounds with AR bioactivity. Most cell-based AR reporter models use transgenic gene constructs that include an androgen response element that controls reporter gene expression. The DNA cis-regulatory elements that respond to AR share sequence similarity with cis-regulatory elements for glucocorticoid (GR, NR3C1), mineralocorticoid (MR, NR3C2), and progesterone (PGR, NR3C3) receptors, which has compromised AR selectivity for some models. In recent years, the sensitivity and selectivity of AR bioassays have been significantly improved through careful selection of cell models, utilization of improved reporter genes, and the use of yeast two-hybrid AR systems. This review summarizes and compares the currently available androgen-responsive cell model systems.

Keywords

androgen receptor - luciferase - GFP - in vitro bioassay - reporter gene assays

• References

• 1 Sar M, Lubahn DB, French FS, Wilson EM. Immunohistochemical localization of the androgen receptor in rat and human tissues. Endocrinology 1990; 127 (6) 3180-3186
• 2 Keller ET, Ershler WB, Chang C. The androgen receptor: a mediator of diverse responses. Front Biosci 1996; 1: d59-71
• 3 Wilson JD, Leihy MW, Shaw G, Renfree MB. Androgen physiology: unsolved problems at the millennium. Mol Cell Endocrinol 2002; 198 (1-2) 1-5
• 4 Raivio T, Tapanainen JS, Kunelius P, Jänne OA. Serum androgen bioactivity during 5alpha-dihydrotestosterone treatment in elderly men. J Androl 2002; 23 (6) 919-921
• 5 Paris F, Servant N, Térouanne B, Sultan C. Evaluation of androgenic bioactivity in human serum by recombinant cell line: preliminary results. Mol Cell Endocrinol 2002; 198 (1-2) 123-129
• 6 Marcelli M, Tilley WD, Zoppi S, Griffin JE, Wilson JD, McPhaul MJ. Androgen resistance associated with a mutation of the androgen receptor at amino acid 772 (Arg----Cys) results from a combination of decreased messenger ribonucleic acid levels and impairment of receptor function. J Clin Endocrinol Metab 1991; 73 (2) 318-325
• 7 Nemoto T, Ohara-Nemoto Y, Shimazaki S, Ota M. Dimerization characteristics of the DNA- and steroid-binding domains of the androgen receptor. J Steroid Biochem Mol Biol 1994; 50 (5-6) 225-233
• 8 Wong CI, Zhou ZX, Sar M, Wilson EM. Steroid requirement for androgen receptor dimerization and DNA binding. Modulation by intramolecular interactions between the NH2-terminal and steroid-binding domains. J Biol Chem 1993; 268 (25) 19004-19012
• 9 Zoppi S, Marcelli M, Deslypere JP, Griffin JE, Wilson JD, McPhaul MJ. Amino acid substitutions in the DNA-binding domain of the human androgen receptor are a frequent cause of receptor-binding positive androgen resistance. Mol Endocrinol 1992; 6 (3) 409-415
• 10 De Vos P, Claessens F, Celis L , et al. Nuclear extracts enhance the interaction of fusion proteins containing the DNA-binding domain of the androgen and glucocorticoid receptor with androgen and glucocorticoid response elements. J Steroid Biochem Mol Biol 1994; 48 (4) 317-323
• 11 Mowszowicz I, Lee HJ, Chen HT , et al. A point mutation in the second zinc finger of the DNA-binding domain of the androgen receptor gene causes complete androgen insensitivity in two siblings with receptor-positive androgen resistance. Mol Endocrinol 1993; 7 (7) 861-869
• 12 Quigley CA, Evans BA, Simental JA , et al. Complete androgen insensitivity due to deletion of exon C of the androgen receptor gene highlights the functional importance of the second zinc finger of the androgen receptor in vivo. Mol Endocrinol 1992; 6 (7) 1103-1112
• 13 Chamberlain NL, Driver ED, Miesfeld RL. The length and location of CAG trinucleotide repeats in the androgen receptor N-terminal domain affect transactivation function. Nucleic Acids Res 1994; 22 (15) 3181-3186
• 14 Simental JA, Sar M, Lane MV, French FS, Wilson EM. Transcriptional activation and nuclear targeting signals of the human androgen receptor. J Biol Chem 1991; 266 (1) 510-518
• 15 van Steensel B, Jenster G, Damm K, Brinkmann AO, van Driel R. Domains of the human androgen receptor and glucocorticoid receptor involved in binding to the nuclear matrix. J Cell Biochem 1995; 57 (3) 465-478
• 16 de Gooyer ME, Deckers GH, Schoonen WG, Verheul HA, Kloosterboer HJ. Receptor profiling and endocrine interactions of tibolone. Steroids 2003; 68 (1) 21-30
• 17 Tomura A, Goto K, Morinaga H , et al. The subnuclear three-dimensional image analysis of androgen receptor fused to green fluorescence protein. J Biol Chem 2001; 276 (30) 28395-28401
• 18 Tyagi RK, Lavrovsky Y, Ahn SC, Song CS, Chatterjee B, Roy AK. Dynamics of intracellular movement and nucleocytoplasmic recycling of the ligand-activated androgen receptor in living cells. Mol Endocrinol 2000; 14 (8) 1162-1174
• 19 Gao W, Bohl CE, Dalton JT. Chemistry and structural biology of androgen receptor. Chem Rev 2005; 105 (9) 3352-3370
• 20 Claessens F, Denayer S, Van Tilborgh N, Kerkhofs S, Helsen C, Haelens A. Diverse roles of androgen receptor (AR) domains in AR-mediated signaling. Nucl Recept Signal 2008; 6: e008
• 21 Moran C, Arriaga M, Arechavaleta-Velasco F, Moran S. Adrenal androgen excess and body mass index in polycystic ovary syndrome. J Clin Endocrinol Metab 2015; 100 (3) 942-950
• 22 Utriainen P, Laakso S, Liimatta J , et al. Premature adrenarche - a common condition with variable presentation. Horm Res Paediatr 2015; 83: 221-231
• 23 Eertmans F, Dhooge W, Stuyvaert S , et al. Endocrine disruptors: effects on male fertility and screening tools for their assessment. Toxicol In Vitro 2003; 17: 515-524
• 24 Cooper ER, McGrath KC, Heather AK. In vitro androgen bioassays as a detection method for designer androgens. Sensors (Basel) 2013; 13 (2) 2148-2163
• 25 Roy P, Alevizaki M, Huhtaniemi I. In vitro bioassays for androgens and their diagnostic applications. Hum Reprod Update 2008; 14 (1) 73-82
• 26 Seeber F, Boothroyd JC. Escherichia coli beta-galactosidase as an in vitro and in vivo reporter enzyme and stable transfection marker in the intracellular protozoan parasite Toxoplasma gondii. Gene 1996; 169 (1) 39-45
• 27 Li XM, Luo FN, Liu GX, Zhu PT. Bioassay of estrogenic activity of effluent and influent in a farm wastewater treatment plant using an in vitro recombinant assay with yeast cells. Biomed Environ Sci 2008; 21 (5) 381-388
• 28 Jiao B, Yeung EK, Chan CB, Cheng CH. Establishment of a transgenic yeast screening system for estrogenicity and identification of the anti-estrogenic activity of malachite green. J Cell Biochem 2008; 105 (6) 1399-1409
• 29 Li J, Li N, Ma M, Giesy JP, Wang Z. In vitro profiling of the endocrine disrupting potency of organochlorine pesticides. Toxicol Lett 2008; 183 (1-3) 65-71
• 30 Möckli N, Auerbach D. Quantitative beta-galactosidase assay suitable for high-throughput applications in the yeast two-hybrid system. Biotechniques 2004; 36 (5) 872-876
• 31 Purvis IJ, Chotai D, Dykes CW , et al. An androgen-inducible expression system for Saccharomyces cerevisiae. Gene 1991; 106 (1) 35-42
• 32 Sohoni P, Sumpter JP. Several environmental oestrogens are also anti-androgens. J Endocrinol 1998; 158 (3) 327-339
• 33 Bitman J, Cecil HC, Harris SJ, Fries GF. Estrogenic activity of o,p'-DDT in the mammalian uterus and avian oviduct. Science 1968; 162 (3851) 371-372
• 34 Kelce WR, Stone CR, Laws SC, Gray LE, Kemppainen JA, Wilson EM. Persistent DDT metabolite p,p'-DDE is a potent androgen receptor antagonist. Nature 1995; 375 (6532) 581-585
• 35 Chatterjee S, Majumder CB, Roy P. Development of a yeast-based assay to determine the (anti)androgenic contaminants from pulp and paper mill effluents in India. Environ Toxicol Pharmacol 2007; 24 (2) 114-121
• 36 Leskinen P, Michelini E, Picard D, Karp M, Virta M. Bioluminescent yeast assays for detecting estrogenic and androgenic activity in different matrices. Chemosphere 2005; 61 (2) 259-266
• 37 Michelini E, Leskinen P, Virta M, Karp M, Roda A. A new recombinant cell-based bioluminescent assay for sensitive androgen-like compound detection. Biosens Bioelectron 2005; 20 (11) 2261-2267
• 38 Lee HJ, Lee YS, Kwon HB , et al. Novel yeast bioassay system for detection of androgenic and antiandrogenic compounds. Toxicol In Vitro 2003; 17: 237-244
• 39 Nishikawa J, Saito K, Goto J, Dakeyama F, Matsuo M, Nishihara T. New screening methods for chemicals with hormonal activities using interaction of nuclear hormone receptor with coactivator. Toxicol Appl Pharmacol 1999; 154 (1) 76-83
• 40 Doesburg P, Kuil CW, Berrevoets CA , et al. Functional in vivo interaction between the amino-terminal, transactivation domain and the ligand binding domain of the androgen receptor. Biochemistry 1997; 36 (5) 1052-1064
• 41 Gaido KW, Leonard LS, Lovell S , et al. Evaluation of chemicals with endocrine modulating activity in a yeast-based steroid hormone receptor gene transcription assay. Toxicol Appl Pharmacol 1997; 143 (1) 205-212
• 42 Xu LC, Sun H, Chen JF , et al. Evaluation of androgen receptor transcriptional activities of bisphenol A, octylphenol and nonylphenol in vitro. Toxicology 2005; 216 (2-3) 197-203
• 43 Zlokarnik G. Fusions to beta-lactamase as a reporter for gene expression in live mammalian cells. Methods Enzymol 2000; 326: 221-244
• 44 Wilkinson JM, Hayes S, Thompson D, Whitney P, Bi K. Compound profiling using a panel of steroid hormone receptor cell-based assays. J Biomol Screen 2008; 13 (8) 755-765
• 45 Sikorski RS, Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 1989; 122 (1) 19-27
• 46 Eldridge ML, Sanseverino J, Layton AC, Easter JP, Schultz TW, Sayler GS. Saccharomyces cerevisiae BLYAS, a new bioluminescent bioreporter for detection of androgenic compounds. Appl Environ Microbiol 2007; 73 (19) 6012-6018
• 47 Térouanne B, Tahiri B, Georget V , et al. A stable prostatic bioluminescent cell line to investigate androgen and antiandrogen effects. Mol Cell Endocrinol 2000; 160 (1-2) 39-49
• 48 Stone KR, Mickey DD, Wunderli H , et al. Isolation of a human prostate carcinoma cell line (DU 145). Int J Cancer 1978; 21: 274-281
• 49 Hartig PC, Bobseine KL, Britt BH , et al. Development of two androgen receptor assays using adenoviral transduction of MMTV-luc reporter and/or hAR for endocrine screening. Toxicol Sci 2002; 66: 82-90
• 50 Kim HJ, Park YI, Dong MS. Comparison of prostate cancer cell lines for androgen receptor-mediated reporter gene assays. Toxicol In Vitro 2006; 20: 1159-1167
• 51 Alimirah F, Chen J, Basrawala Z, Xin H, Choubey D. DU-145 and PC-3 human prostate cancer cell lines express androgen receptor: implications for the androgen receptor functions and regulation. FEBS Lett 2006; 580 (9) 2294-2300
• 52 Veldscholte J, Berrevoets CA, Ris-Stalpers C , et al. The androgen receptor in LNCaP cells contains a mutation in the ligand binding domain which affects steroid binding characteristics and response to antiandrogens. J Steroid Biochem Mol Biol 1992; 41 (3-8) 665-669
• 53 Sun H, Xu XL, Xu LC , et al. Antiandrogenic activity of pyrethroid pesticides and their metabolite in reporter gene assay. Chemosphere 2007; 66 (3) 474-479
• 54 Vinggaard AM, Joergensen EC, Larsen JC. Rapid and sensitive reporter gene assays for detection of antiandrogenic and estrogenic effects of environmental chemicals. Toxicol Appl Pharmacol 1999; 155 (2) 150-160
• 55 Roy P, Salminen H, Koskimies P , et al. Screening of some anti-androgenic endocrine disruptors using a recombinant cell-based in vitro bioassay. J Steroid Biochem Mol Biol 2004; 88 (2) 157-166
• 56 Blankvoort BM, de Groene EM, van Meeteren-Kreikamp AP, Witkamp RF, Rodenburg RJ, Aarts JM. Development of an androgen reporter gene assay (AR-LUX) utilizing a human cell line with an endogenously regulated androgen receptor. Anal Biochem 2001; 298 (1) 93-102
• 57 Wilson VS, Bobseine K, Lambright CR , et al. A novel cell line, MDA-kb2, that stably expresses an androgen- and glucocorticoid-responsive reporter for the detection of hormone receptor agonists and antagonists. Toxicol Sci 2002; 66: 69-81
• 58 Sonneveld E, Jansen HJ, Riteco JA , et al. Development of androgen- and estrogen-responsive bioassays, members of a panel of human cell line-based highly selective steroid-responsive bioassays. Toxicol Sci 2005; 83: 136-148
• 59 Sedlák D, Paguio A, Bartůněk P. Two panels of steroid receptor luciferase reporter cell lines for compound profiling. Comb Chem High Throughput Screen 2011; 14 (4) 248-266
• 60 Paguio A, Stecha P, Wood KV , et al. Improved dual-luciferase reporter assays for nuclear receptors. Curr Chemical Genomics 2010; 4: 43-49
• 61 March JC, Rao G, Bentley WE. Biotechnological applications of green fluorescent protein. Appl Microbiol Biotechnol 2003; 62 (4) 303-315
• 62 Bovee TF, Helsdingen RJ, Koks PD, Kuiper HA, Hoogenboom RL, Keijer J. Development of a rapid yeast estrogen bioassay, based on the expression of green fluorescent protein. Gene 2004; 325: 187-200
• 63 Lyttle CR, Damian-Matsumura P, Juul H, Butt TR. Human estrogen receptor regulation in a yeast model system and studies on receptor agonists and antagonists. J Steroid Biochem Mol Biol 1992; 42 (7) 677-685
• 64 Xu H, Kraus WL, Shuler ML. Development of a stable dual cell-line GFP expression system to study estrogenic endocrine disruptors. Biotechnol Bioeng 2008; 101 (6) 1276-1287
• 65 Bovee TF, Lommerse JP, Peijnenburg AA, Fernandes EA, Nielen MW. A new highly androgen specific yeast biosensor, enabling optimisation of (Q)SAR model approaches. J Steroid Biochem Mol Biol 2008; 108 (1-2) 121-131
• 66 Beck V, Reiter E, Jungbauer A. Androgen receptor transactivation assay using green fluorescent protein as a reporter. Anal Biochem 2008; 373 (2) 263-271
• 67 Dennis MK, Bowles HJ, MacKenzie DA , et al. A multifunctional androgen receptor screening assay using the high-throughput Hypercyt flow cytometry system. Cytometry A 2008; 73: 390-399
• 68 Marcelli M, Stenoien DL, Szafran AT , et al. Quantifying effects of ligands on androgen receptor nuclear translocation, intranuclear dynamics, and solubility. J Cell Biochem 2006; 98 (4) 770-788
• 69 Nazareth LV, Stenoien DL, Bingman III WE , et al. A C619Y mutation in the human androgen receptor causes inactivation and mislocalization of the receptor with concomitant sequestration of SRC-1 (steroid receptor coactivator 1). Mol Endocrinol 1999; 13 (12) 2065-2075
• 70 Szafran AT, Szwarc M, Marcelli M, Mancini MA. Androgen receptor functional analyses by high throughput imaging: determination of ligand, cell cycle, and mutation-specific effects. PLoS ONE 2008; 3 (11) e3605
• 71 Veldscholte J, Ris-Stalpers C, Kuiper GG , et al. A mutation in the ligand binding domain of the androgen receptor of human LNCaP cells affects steroid binding characteristics and response to anti-androgens. Biochem Biophys Res Commun 1990; 173 (2) 534-540
• 72 Marcelli M, Zoppi S, Wilson CM, Griffin JE, McPhaul MJ. Amino acid substitutions in the hormone-binding domain of the human androgen receptor alter the stability of the hormone receptor complex. J Clin Invest 1994; 94 (4) 1642-1650