Androgens are widely acknowledged to be central to the pathogensis of benign prostatic hypertrophy (BPH). However (GIBCO) with 5% fetal bovine serum containing penicillin (100 units/ml), streptomycin sulfate (100 ,ug/ml), and amphotericin (0.25 gg/ml) and was incubated for 2-3 days. It was then minced into 1-mm3 portions and transferred to 16-mm wells in serum-free medium (0.5 ml of RPMI 1640) for about 18 hr before the start of an experiment. All additions (SHBG, steroids, controls) were made in serum free RPMI 1640.Cell culture. Fibroblast-enriched cultures (17) were prepared from surgical specimens obtained as above. Fragments (1-3 mm3) were placed in 25 ml of digestion buffer (10 mM Hepes/142 mM NaCl/6.7 mM KC1, pH 7.4) and stirred for 30 min at 370C in an atmosphere of 95% air/5% CO2. After discarding the supernatant, fresh digestion buffer supplemented with 1% collagenase, 0.67 mM CaCl2, 20 mM dextrose, and 0.05% DNase was added to the tissue for 30 min at 370C. The supernatant was discarded again and the digestion was repeated twice. The cells released after the second and third digestion were collected, washed twice, and plated in fresh attachment medium (Ham's F12 supplemented with 5% fetal bovine serum). The medium was changed after 7 days and every 3-4 days thereafter. Three to four weeks after the original isolation, >95% of the cells had the typical morphology of fibroblasts. About 18 hr before the beginning of an experiment, cells were placed in serum-free medium (Ham's F12), and detached with a nonenzymatic cell dissociation solution (Sigma). They then were washed once and suspended (0.2-0.5 mg of protein per ml) in serum-free medium containing 50 nM SHBG. Prostatic epithelial cells were provided by D. M. Peehl (Stanford University) and were isolated as described by her (18). They underwent the same experimental protocol as the fibroblasts.Abbreviations: BPH, benign prostatic hypertrophy; SHBG, sex hormone-binding globulin.
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