Han-Jung Lee¹, Irene Mowszowicz², and Chawnshang Chang¹

¹Department of Medicine, and Endocrinology-Reproductive Physiology Program, University of Wisconsin, Comprehensive Cancer Center, Madison, WI 53792

²Biochemistry Laboratory B, Hospital Necker, Paris 75743, France

Received 04/19/96; Accepted 06/10/96; On-line 07/01/96

Cell culture.

Fibroblast strains were obtained from skin biopsies of patients with AIS and propagated in a 4/1 mixture of medium 199/Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics and 10% fetal calf serum as previously described (8).

Androgen-binding capacity.

Androgen-binding capacity was measured as previously described (9) on intact monolayers grown to confluency in 100 mm petri dishes. Replicate culture received increasing concentrations (0.05-2.0 nM) of [³H]-5-dihydrotestosterone (DHT) in a serum-free medium, alone or with two-hundred-fold excess of non-radioactive DHT. Cells were incubated for 60 min at 37°C under normal culture conditions. At the end of incubation, cells were washed, harvested and nuclear proteins were extracted by sonication in 50 mM Tris-HCl, pH 7.4 buffer containing 1 mM EDTA and 0.6 M KCl. Free and loosely bound DHT was removed by dextran-coated charcoal treatment. Specific binding was calculated as the difference between total binding ([³H]-DHT alone) and nonspecific binding (nondisplaced in the presence of an excess of unlabeled DHT). The maximum binding capacity (B_max) and the apparent dissociation constant (K_D) of the androgen receptor were derived from Scatchard plot analysis. Binding capacity was expressed as femtomoles of DHT bound per mg DNA. For each patient, binding capacity was measured at least twice in separate experiments. In some experiments, incubations were continued for 18 h to assess up-regulation of the AR. These conditions usually result in a 2- to 3-fold increase of the normal AR (10). DNA was assayed after perchloric acid extraction as previously described (11).

5alpha-Reductase enzyme assay.

5alpha-Reductase activity was measured as previously described (9). Cells were cultured in 60 mm Petri dishes containing serum-free medium 24 h before assay. The medium was replaced by 2 ml of serum-free medium containing 5 nM [³H]-testosterone (50 to 60 Ci/mmol) and incubated for 15 min to 2 h (at least 3 time points) at 37°C in 5% CO₂. The dishes were then put on ice to stop the reaction. Fifty µl of medium from each dish was added into counting vials to measure the total radioactivity. To monitor recovery, the medium from individual dish was transferred into a tube containing [¹⁴C]-tracers (10,000 cpm of testosterone, 1,000 cpm of DHT, 1,000 cpm of 3alpha- or 3ß-Diol, and 1,000 cpm of androstenedione). The medium from each dish was transferred into another tube containing unlabeled tracers (4 mg/ml ethanol), and extracted by vortexing 1 min with 10 ml of ethyl acetate/cyclohexane 1/1 (v/v). The organic phase was evaporated to dryness and analyzed by thin-layer chromatography in chloroform/methanol 97.5/2.5 (v/v) for 90 min. Testosterone and androstenedione were visualized under UV light (240 nm) and 5alpha-reduced steroids in iodine vapors. Steroids were scraped from the silica gel plates (Merck, GF254), and eluted in acetone and ethanol. 3alpha- and 3ß-Diols, not separated in this system, were eluted together. Eluates were evaporated to dryness and counted for radioactivity. 5alpha-Reductase activity was calculated as the amount of DHT and Diols formed in 1 h and expressed as fmol/µg DNA/h.

RNA extraction.

Cells (6 x 10⁶) grown to confluency and placed in serum-free medium 24 h before extraction were harvested using trypsin-EDTA, collected in serum-free medium, and centrifuged at 200 x g for 15 min. The cell pellet was washed 3 times with sterile saline and transferred into two eppendorf tubes (1.5 ml). The cell pellet was either extracted immediately or stored at -80° C. Extraction was performed using the one-step RNAzol extraction procedure (12). The cell pellet was carefully suspended in 600µl of RNAzol (Bioprobe Systems, France) and 60µl chloroform/isoamylic alcohol (24/1) was added. Tubes were vigorously agitated, allowed to stand 15 min on ice, and centrifuged at 11,750 x g, 2° C for 15 min. The aqueous phase was transferred into a clean eppendorf tube and RNA was precipitated in isopropanol for 2 h at -80° C.

Complementary DNA preparation.

The following primers were used for separate analysis of the N-terminal domain (NTD), DNA-binding domain (DBD), and ligand-binding domain (LBD), which cover the entire AR cDNA (6, 7).

primer 5 of NTD: 5'-GTGCAGTTAGGGCTGGGAAGG-3'

primer G of NTD: 5'-CGGGTTCTCCAGCTTGATGCG-3'

primer 2 of DBD: 5'-TCGCGACTACTACAACTTTC-3'

primer 4 of DBD: 5'-TGGCTCAATGGCTTCCAGGA-3'

primer A of LBD: 5'- GTGGAAATAGATGGGCTTGA-3'

primer B of LBD: 5'-TCACACATTGAAGGCTATGA-3'

Complementary DNAs were synthesized using 1µg of total cellular RNA by the GeneAmp RNA PCR Kit (Perkin Elmer, Branchburg, NJ) according to the manufacturer's specifications. Amplification by polymerase chain reaction (PCR) was performed using two-thirds of the cDNA synthesis reaction mixture and the above pairs of primers (100 ng each) in a total volume of 100µl by Perkin Elmer PCR apparatus during 30 cycles. Each cycle included denaturation for 1 min at 95° C, annealing for 2 min at 60° C, and extension for 3 min at 72° C. After the last cycle, samples were incubated at 72° C for 7 min. The amplified products were recovered after phenol/chloroform extraction and ethanol precipitation.

DNA cloning and sequencing.

Amplified cDNAs were cloned by either the TA (InVitrogen, San Diego, CA) or pT7Blue cloning system (Novagen, Madison, WI) as described in the manufacturer's instruction. Sequence was analyzed by the dideoxynucleotide chain termination method (13) in SequaGel system (National Diagnostics, Manville, NJ) directly on plasmid DNA (14).