5α-Reductase in Intact DU145 Cells
203
(5′-AAA AGA TGA ATG GAA TAA GGG CTT TCC G-3′). All primers
were synthesized by MWG-Biotech (Ebersberg, Germany). PCR was per-
formed with nucleic acid extracts from DU145 cells with or without RNase
A treatment. PCR conditions (thermocycler primus 25, MWG-Biotech) were
1 min at 94 °C (denaturation), 2 min at 64 °C (anealing) and 3 min at 72 °C
(elongation). As controls the plasmids ph5α45 and pBS-76-1 were used as
templates. Plasmid ph5α45 contains a full-length human cDNA encoding
5α-reductase type I and plasmid pBS-76-1 contains a full-length human
cDNA encoding 5α-reductase type II. Temperature gradient PCR was per-
formed using a Mastercycler gradient apparatus (Eppendorf, Köln, Ger-
many).
The PCR-amplified fragment encoding type I 5α-reductase was ligated
into the pks (+)-cloning vector (Stratagene, Groningen, Netherlands) and
submitted to DNA sequence analysis which was performed by SEQLAB
(Göttingen, Germany).
Inhibition Assay
DU145 cells were used as source of 5α-reductase type I isozyme between
passages 15–40. Cells were plated out one day before the experiment in
24-well plates at a density of 1.6 × 105 cells/well. DU145 cells were allowed
to become adherent overnight in 1 ml RPMI-1640 medium (with 10% FCS).
Appropriate concentrations (10 µM final concentration at initial tests) of
inhibitors dissolved in dimethyl sulfoxide (DMSO) were applied in dupli-
cates. Growth medium was replaced by 500 µl of fresh medium containing
5 µl of the inhibitor and 5 nM [3H]androstenedione as substrate. At least 3
controls were performed with 5 µl DMSO (without inhibitor) and 495 µl
RPMI (10% FCS) containing the substrate. After a 6 h incubation period (5%
CO2 in a humidified atmosphere at 37 °C), the medium was again replaced
and extracted with 800 µl diethyl ether. Steroids were dried by evaporation
of the organic phase. After resuspension in 50 µl ethanol steroid metabolites
were subjected to HPLC analysis. Results are expressed as the amount of
androstanedione produced as a percentage of control values. Compounds
showing more than 70%inhibitionof thesubstrateconversionrate(compared
to the uninhibited controls) were subsequently tested at three different
concentrations for the determination of IC50 values.
Reversed Phase HPLC
HPLC analyses were performed by the use of a high pressure solvent
delivery pump (Waters M6000A, Milford, USA), a radioactivity detector
(LB506C, Berthold, Wildbad, Germany) and an autosampler system (851-
AS, Jasco, Tokyo, Japan). Nucleosil 120-3-C8 was applied as stationary
phase using prepacked columns (125×4 mm; Macherey-Nagel, Düren, Ger-
many). The injection volume was 13 µl and methanol/water (50/50) was used
as the mobile phase for separation of the steroidal metabolites. Applying a
flow rate of 0.4 ml/min, the retention times were as follows: androstenedione:
11.2 min, androstanedione: 17.5 min. Data acquisition and integration were
carried out by the use of the HALABE 1.6.5 software (Berthold, Wildbad,
Germany).
Figure 4. Structures of new 5α-reductase type I inhibitors identified using
the DU145 whole cell assay.
Experimental
Cell Culture
Test Compounds
The human prostatic carcinoma cell line DU145 (DSM ACC 261) was
obtained from the DSMZ (German collection of microorganisms and cell
cultures, Braunschweig, Germany). The cells were maintained as a mono-
layer culture in RPMI-1640 medium. The medium was supplemented with
10% (v/v) fetal calf serum (FCS), 0.25% sodium bicarbonate, 100 units
penicillin/ml and 100 µg streptomycin/ml. Cells were grown in 175 cm2
tissue culture flasks (Nunc, Wiesbaden, Germany) in a humidified 95% O2
and 5% CO2 atmosphere at 37 °C. The cell line was routinely passaged every
4 days after treatment with 0.25% trypsin solution. All tissue culture reagents
were obtained from c.c.pro (Neustadt/W., Germany).
The new 5α-reductase inhibitors have been synthesized in our group:
EB18, EB25, EB33 and EB34 [15], EB40 [16], MH65 [17], and MK77 [18]
.
References
✩
Dedicated to Prof. H. Becker on the occasion of his 60th birthday.
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PCR and Sequence Analysis
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The following primers were used for PCR experiments: WOR1 (5′-CCC
AAG CTT GGG ACC ATG GCA ACG GCG ACG GGG GTG GCG G-3′),
WOR2 (5′-AAA GTC CAT AGA GAA GCG CCA TTG G-3′), WOR6
(5′-GAA CAC GGC GCG ATG CAG GTT CAG TGC CAG–3′) and WOR7
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[4] J. D. Wilson, Handb. Physiol. 1975, 5, 491–508.
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