Expression and Characterization of Recombinant Type 2 3
-Hydroxysteroid Dehydrogenase (HSD) from Human Prostate: Demonstration of Bifunctional 3
/17ß-HSD Activity and Cellular Distribution
Hsueh-Kung Lin1,
Joseph M. Jez1,
Brian P. Schlegel,
Donna M. Peehl,
Jonathan A. Pachter and
Trevor M. Penning
Departments of Pharmacology, Biochemistry, and Biophysics (H.K.L.,
J.M.J., B.P.S., T.M.P) University of Pennsylvania School of
Medicine Philadelphia, Pennsylvania 19104
Department of
Urology (D.M.P) Stanford University Medical School Stanford,
California 94305
Schering-Plough Research Institute
(J.A.P.) Kenilworth, New Jersey 07033
 |
ABSTRACT
|
---|
In androgen target tissues, 3
-hydroxysteroid
dehydrogenase (3
-HSD) may regulate occupancy of the androgen
receptor (AR) by catalyzing the interconversion of
5
-dihydrotestosterone (5
-DHT) (a potent androgen) and
3
-androstanediol (a weak androgen). In this study, a 3
-HSD cDNA
(1170 bp) was isolated from a human prostate cDNA library. The human
prostatic 3
-HSD cDNA encodes a 323-amino acid protein with 69.9%,
84.1%, 99.4%, and 87.9% sequence identity to rat liver 3
-HSD and
human type 1, type 2, and type 3 3
-HSDs, respectively, and is a
member of the aldo-keto reductase superfamily. The close homology with
human type 2 3
-HSD suggests that it is either identical to this
enzyme or a structural allele. Surprisingly, when the recombinant
protein was expressed and purified from Escherichia coli,
the enzyme did not oxidize androsterone when measured
spectrophotometrically, an activity previously assigned to recombinant
type 2 3
-HSD using this assay. Complete kinetic characterization of
the purified protein using spectrophotometric, fluorometric, and
radiometric assays showed that the catalytic efficiency favored
3
-androstanediol oxidation over 5
-DHT reduction. Using
[14C]-5
-DHT as substrate, TLC analysis
confirmed that the reaction product was
[14C]-3
-androstanediol. However, in the
reverse reaction, [3H]-3
-androstanediol
was oxidized first to [3H]-androsterone and
then to [3H]-androstanedione, revealing that
the expressed protein possessed both 3
- and 17ß-HSD activities.
The 17ß-HSD activity accounted for the higher catalytic efficiency
observed with 3
-androstanediol. These findings indicate that, in the
prostate, type 2 3
-HSD does not interconvert 5
-DHT and
3
-androstanediol but inactivates 5
-DHT through its 3-ketosteroid
reductase activity. Levels of 3
-HSD mRNA were measured in primary
cultures of human prostatic cells and were higher in epithelial cells
than stromal cells. In addition, elevated levels of 3
-HSD mRNA were
observed in epithelial cells derived from benign prostatic hyperplasia
and prostate carcinoma tissues. Expression of 3
-HSD was not prostate
specific, since high levels of mRNA were also found in liver, small
intestine, colon, lung, and kidney. This study is the first complete
characterization of recombinant type 2 3
-HSD demonstrating dual
activity and cellular distribution in the human prostate.
 |
INTRODUCTION
|
---|
Testosterone, after entering prostatic cells, is translocated to
the nucleus and converted to 5
-dihydrotestosterone (5
-DHT) by
3-oxo-5
-steroid-4-dehy-drogenase (5
-reductase) (1, 2).
5
-DHT2 is a more potent
androgen than testosterone in stimulating prostate growth (3, 4) and
preferentially binds to the androgen receptor [AR; dissociation
constant (Kd) for the AR of 10-11
M] (5, 6). Elevation of 5
-DHT content in prostate has
been associated with benign prostatic hyperplasia (BPH) in humans and
canines (5, 6, 7, 8, 9) and with human prostate carcinoma (PCA) (10, 11). The
action of 5
-DHT may be terminated by 3
-HSD, which catalyzes the
formation of 3
-androstanediol (a weak androgen; Kd for
the AR of 10-6 M) (Fig. 1
) (12). It has also
been proposed that, by catalyzing the reverse reaction, 3
-HSD may
function as a molecular switch and, in this manner, may regulate the
amount of 5
-DHT available for AR binding and activation.
Detailed studies of 3
-HSD in prostate are required to understand its
role in androgen metabolism and action. First, controversy exists over
the directionality of the 3
-HSD reaction in the prostate. Studies in
dog (13, 14) and rat (15) prostate indicate that the dehydrogenase
activity of 3
-HSD is favored over the reductase activity; others
suggest that the opposite occurs in the rat (16, 17), dog (18), and
human (19) prostate. Kinetic characterization of recombinant 3
-HSD
obtained from human prostate should help clarify these issues. Second,
it is uncertain whether 3
-HSD is localized to the same cells as
5
-reductase and the AR in the prostate, and this may impact on its
ability to regulate occupancy of the AR.
Attempts to characterize 3
-HSD from rat, dog, and human prostate
have been hampered by difficulties in obtaining pure protein. Partially
purified 3
-HSD from rat prostate shares properties in common with
the rat liver enzyme: it functions as an oxidoreductase with dual
pyridine nucleotide specificity acting on both 5
- and 5ß-reduced
steroids (16, 17). In dog and human prostate, cytosolic and microsomal
3
-HSD activities have been described (20, 21, 22). Recently, 3
-HSD
from human hyperplastic prostate has been partially purified (23).
However, no homogeneous 3
-HSD expressed in human prostate has been
obtained in significant amounts from any source.
3
-HSD of the human prostate may be similar to the extensively
studied 3
-HSD from rat liver (24). The rat liver 3
-HSD
(AKR1C9)3 cDNA has been cloned (26) and is
a member of the aldo-keto reductase (AKR) superfamily that includes
human type 1 (AKR1C4), type 2 (AKR1C3), and type 3 (AKR1C2) 3
-HSDs
(27, 28). Recombinant rat liver 3
-HSD has been overexpressed in
E. coli and shown to be kinetically identical to protein
purified directly from rat liver (29). Also, the three-dimensional
structures of rat liver 3
-HSD apoenzyme, the ENADP+
binary complex, and E · NADP+ · testosterone
ternary complex have been solved by x-ray crystallography (30, 31, 32) and
site-directed mutagenesis studies performed (29, 33).
To investigate the potential role of 3
-HSD in the regulation of
androgen levels in human prostate, we have cloned a cDNA virtually
identical to type 2 3
-HSD from a human prostate library. This work
represents the first complete kinetic characterization of a homogenous
3
-HSD expressed in human prostate. Our results demonstrate that this
protein is better described as a 3
/17ß-HSD, and that this enzyme
may remove 5
-DHT from the prostatic androgen pool through its
3-ketosteroid reductase activity. Examination of mRNA levels in primary
cultures of prostatic cells derived from normal, BPH, and PCA tissues
reveals that the transcript is expressed predominantly in epithelial
cells [where the AR is found in the absence of type 2 5
-reductase
(34)] suggesting a possible role for 3
-HSD in terminating androgen
action. Importantly, 3
-HSD mRNA expression may be elevated in both
BPH and PCA.
 |
RESULTS
|
---|
Cloning and Sequencing of a cDNA Encoding Type 2 3
-HSD from
Human Prostate
A human colon dihydrodiol dehydrogenase (DD) 1 (AKR1C1) cDNA probe
was used to screen a human prostate cDNA library. Although DD1 is
predominantly a 20
-HSD, it differs from DD2 (type 3 3
-HSD and
bile acid-binding protein) by only seven amino acids (35, 36). The
initial screen detected 13 positive clones. Partial sequences derived
from these clones shared similar, but not identical, nucleotide
sequences (data not shown). The complete sequence of one of these
clones that possessed the highest sequence identity with the probe was
acquired, but there was no ATG translation start codon, indicating that
the clone was not full length. Subsequent rapid amplification of
5'-cDNA ends (5'-RACE) identified the ATG start site and two missing
codons. The 5'-RACE sequence is shown in Fig. 2
, and the complete sequence of the cDNA
is shown in Fig. 3
. The cDNA clone of
1170 bp encodes a protein of 323 amino acids with a calculated
molecular mass of 36,815 daltons and contained a 3'-untranslated
region (UTR) and a polyadenylation signal.

View larger version (16K):
[in this window]
[in a new window]
|
Figure 2. Identification of the 5'-UTR Region of Human Type 2
3 -HSD Using 5'-RACE
A, Gel electrophoresis of 5'-RACE product. The amplified DNA product
was separated on a 1.2% agarose gel and stained with ethidium bromide.
One single band with size of 650 bp was preferentially amplified. B,
Nucleotide sequence of the amplified DNA product from three randomly
selected plasmids. All three extended products contained identical
sequence in the coding region, but the length of their 5'-UTRs
differed. This may result from either different individuals that are
present in the pooled poly(A)+ RNA template, alternate
transcription start sites for type 2 3 -HSD, or an artifact of
immature reverse transcription. Sequences upstream of the ATG start
sites are shown; sequences downstream from the ATG start site are
underlined.
|
|

View larger version (61K):
[in this window]
[in a new window]
|
Figure 3. Nucleotide Sequence and the Predicted Amino Acid
Sequence of the Human Prostate 3 -HSD Clone
The open reading frame contains 969 nucleotides and encodes a protein
of 323 amino acids. The polyadenylation site is
underlined.
|
|
Comparison of the predicted amino acid sequence of the cDNA clone with
members of the AKR superfamily showed that the protein was 99.4%
identical to human type 2 3
-HSD (AKR1C3; Ref.27) differing only at
residues 75 and 175. In addition, this clone has an identical 5'-UTR
and shares 94.7% identity with the 3'-UTR of human type 2 3
-HSD. On
this basis it was assumed that the human prostatic 3
-HSD is type 2
3
-HSD. The coding region of the prostatic HSD clone also shared
69.9%, 84.1%, 87.9%, and 87.0% amino acid identity with rat liver
3
-HSD (AKR1C9; Ref.29), human type 1 3
-HSD (AKR 1C4; Ref.27),
human type 3 3
-HSD (AKR1C2; Ref.28), and human DD1 (AKR1C1; Refs.
35 and 36), respectively (Fig. 4
).
Overexpression and Purification of Human Recombinant Type 2
3
-HSD
To identify the enzymatic activity of the protein encoded by the
cDNA clone, a prokaryotic expression construct was generated and used
to transform E. coli cells. Surprisingly, E. coli
sonicates expressing the recombinant protein failed to oxidize
androsterone using a spectrophotometric assay, an activity previously
assigned to recombinant type 2 3
-HSD using this assay (27). This
result questioned either the identity of the clone or the substrate
specificity of human type 2 3
-HSD. To completely characterize the
enzyme activity of the recombinant protein, it was purified to
homogeneity in a three-step process to yield 1015 mg. Since the
enzyme was unable to oxidize androsterone, the purification was
followed by SDS-PAGE (Fig. 5A
). The
homogeneous enzyme gave a single band on SDS-PAGE similar in size to
the 3
-HSD partially purified from human and rat prostate (16, 23).
The protein was also immunoreactive to a rabbit anti-rat liver 3
-HSD
antiserum (Fig. 5B
).

View larger version (45K):
[in this window]
[in a new window]
|
Figure 5. Expression and Purification of Recombinant
Prostatic Type 2 3 -HSD from E. coli Cells
A, SDS/PAGE analysis: lane 1, 5 µg purified recombinant rat
liver 3 -HSD; lane 2, 30 µg bacterial cell sonicate; lane 3, 15
µg of the peak fraction from a DE52 cellulose column; lane 4, 5 µg
of the peak fraction from a Blue Sepharose affinity column (final
step). B, Western blot analysis: lane 1, 0.3 µg purified recombinant
rat liver 3 -HSD; lane 2, 5 µg bacterial cell sonicate; lane 3, 1
µg of the cellulose column peak fraction; lane 4, 0.2 µg of the
Blue Sepharose purified protein. Molecular mass markers for panels A
and B are noted in kilodaltons.
|
|
To confirm proper folding of the overexpressed human type 2 3
-HSD,
the binding of NADPH was measured by fluorescence titration. Based on
the three-dimensional structure of rat liver 3
-HSD complexed with
cofactor (31, 32), the nicotinamide-binding pocket contains 13
different amino acids encompassing a large portion of the
(
/ß)8-barrel structure and binds NADPH in an unusual
extended conformation with a Kd = 143 nM (29).
Any significant alterations in folding of the overexpressed protein
would alter the binding affinity for NADPH. In type 2 3
-HSD, nine of
these residues are invariant, and the remaining four substitutions are
conservative ones. Titration of type 2 3
-HSD with NADPH yielded a
calculated Kd of 107 ± 8 nM, indicating
that the purified protein was properly folded. Therefore, the inability
to oxidize androsterone can not be attributed to either poor protein
expression or the expression of improperly folded protein.
Kinetic Characterization and Product Identification of Recombinant
Human Type 2 3
-HSD
Characterization of the homogeneous recombinant protein initially
focused on obtaining specific activities for a variety of substrates at
different pH values using NAD(H) and NADP(H) as cofactors. Our results
suggested that the purified protein had 3
-HSD activity, oxidizing
3
-androstanediol and reducing 5
-DHT and androstanedione (Table 1
). The recombinant enzyme displayed no
detectable 17ß- or 20
-HSD activity using the standard
spectrophotometric method. As in other HSDs of the AKR superfamily,
dual pyridine-nucleotide specificity was observed. Also, the purified
protein had similar pH preferences as rat liver 3
-HSD: at pH 6.0,
the reduction reaction rate increased; and at pH 9.0, the oxidation
reaction rate increased.
Kinetic constants for the physiological substrates 5
-DHT,
3
-androstanediol, and androstanedione were determined
spectrophotometrically. Based on these assays, comparison of
kcat/Km values suggested that type 2 3
-HSD
favors oxidation of 3
-androstanediol over reduction of 5
-DHT
(Table 2
). In contrast, the reduction of
androstanedione is more efficient than oxidation of androsterone, which
was barely detectable (Table 2
). For comparative purposes, we also used
compounds (i.e. 9,10-phenanthrenequinone and
4-nitrobenzaldehyde) that are common substrates for other members of
the AKR superfamily. These ubiquitous AKR substrates, while efficiently
turned over, are nonphysiological. Human prostatic type 2 3
-HSD had
a 20-fold higher Km and a slightly lower Vmax
for 9,10-phenanthrenequinone than the rat liver enzyme, whereas the
human prostate and rat liver enzyme gave similar
kcat/Km values for 4-nitrobenzaldehyde (Table 2
).
The low turnover numbers for endogenous androgen substrates
necessitated the use of fluorometric and radiometric assays. By
monitoring the fluorescence emission of NADPH at 450 nm or the
disappearance of radioactive steroid by TLC, these methods
increased sensitivity 100- or 2500-fold, respectively, over the
standard spectrophotometric assay. The kinetic constants obtained by
these methods were in good agreement with values estimated by the
spectrophotometric assays (Table 3
).
The radiometric assay showed that, as expected,
[14C]-5
-DHT was converted into
[14C]-3
-androstanediol by type 2 3
-HSD,
confirming its ability to reduce 3-ketosteroids (Fig. 6A
). However, incubation of type 2
3
-HSD with [3H]-3
-androstanediol did not produce
[3H]-5
-DHT. Instead, androsterone was produced that
was subsequently converted to androstanedione (Fig. 6B
). These results
reveal for the first time that type 2 3
-HSD also displays 17ß-HSD
activity.

View larger version (58K):
[in this window]
[in a new window]
|
Figure 6. Radiochemical Assay and Product Identification
A, Conversion of 5 -DHT into 3 -androstanediol: lane 1, incubation
of 5 -DHT without enzyme; lane 2, incubation with recombinant rat
liver 3 -HSD (15 min with 3 µg); lane 3, incubation with
recombinant human type 2 3 -HSD (60 min with 15 µg). B, Conversion
of 3 -androstanediol into androsterone and androstanedione: lane 1,
incubation of 3 -androstanediol without enzyme; lane 2, incubation
with recombinant rat liver 3 -HSD (15 min with 3 µg); lanes 3, 4,
5, and 6, incubation with recombinant human type 2 3 -HSD (15 µg)
for 15, 30, 45, and 60 min, respectively; lane 7, incubation of
androsterone without enzyme; lane 8, incubation of androsterone with
recombinant rat liver 3 -HSD (15 min with 3 µg).
|
|
Since androsterone was formed before androstanedione appeared, it
seems that the 17ß-HSD activity is favored over 3
-HSD activity in
the oxidation direction. Further characterization of the 17ß-HSD
activity used [14C]-5
-DHT and
[14C]-testosterone as substrates for oxidation (Table 3
).
Comparison of the catalytic efficiencies for these reactions with that
for [3H]-androsterone oxidation indicated that type 2
3
-HSD preferentially oxidizes 17ß-hydroxysteroids. Similarly,
comparison of the catalytic efficiency for the oxidation of
[3H]-3
-androstanediol with that for
[3H]-androsterone shows that the 17ß-alcohol is
preferentially oxidized over the 3
-alcohol of the diol. Thus, the
higher catalytic efficiency observed with 3
-androstanediol over
5
-DHT is primarily due to the 17ß-HSD activity of the enzyme.
These data suggest that, in the prostate, type 2 3
-HSD does not
interconvert 5
-DHT with 3
-androstanediol but functions to
inactivate 5
-DHT as a result of its 3-ketosteroid reductase
activity.
Northern Blot Analysis of 3
-HSD in Primary Cultures of Human
Prostatic Cells
Since type 2 3
-HSD can inactivate the potent androgen 5
-DHT,
the distribution of 3
-HSD mRNA was examined within the prostate.
Northern analysis was used to detect the levels of mRNA in primary
cultures of epithelial and stromal cells derived from normal, BPH, and
PCA prostate tissues. A 1.2-kb 3
-HSD transcript was predominantly
expressed in epithelial cells (Fig. 7
).
Remarkably, there was evidence for elevated expression in the
epithelial cells derived from areas of the prostate with either BPH and
PCA histology. The gels contained similar amounts of RNA as judged by
levels of 28S and 18S rRNA, and the transfer was equivalent across the
lanes based on hybridization with a probe for human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Since type 2 3
-HSD
shares high sequence homology with other AKR superfamily members, which
may affect the specificity of hybridization, the Northern blots were
stripped and reprobed with the 3'-UTR whose sequence is closely related
only to other 3
-HSD isoforms of the AKR superfamily. Using the
3'-UTR probe, the presence of 3
-HSD mRNAs was primarily limited to
epithelial cells and was elevated in cells from diseased prostatic
tissues. Quantification of the Northern analysis using the 3'-UTR probe
showed that the ratios of 3
-HSD:GAPDH mRNA were 0.48 ± 0.08,
0.77 ± 0.25, and 1.60 ± 0.57 for epithelial cells derived
from normal, BPH, and PCA tissues, respectively. Based on Students
t test in which P < 0.05 indicates
significance, the difference in expression levels was statistically
significant between cells derived from normal and PCA tissues.
Tissue-Specific Expression
Since type 2 3
-HSD was originally cloned from human liver (27),
its expression is not prostate specific. To examine the distribution of
this enzyme, Northern analysis was performed on RNA from 16 different
human tissues (Fig. 8
). In each tissue, a
single 1.2-kb mRNA species was observed using the 3'-UTR for type 2
3
-HSD as a probe (see above). The levels of 3
-HSD mRNA were
highest in liver, colon, and small intestine. The spleen, placenta, and
brain had the lowest levels of 3
-HSD transcript. Due to the
specificity of the probe, the levels of mRNA detected probably reflect
the total pool of 3
-HSD isoforms in each tissue.

View larger version (83K):
[in this window]
[in a new window]
|
Figure 8. Tissue Distribution of 3 -HSD in Human Tissues
Northern blots containing RNA from 16 different human tissues were
hybridized with a randomly primed probe corresponding to human type 2
3 -HSD 3'-UTR (+995 to +1170 bp) and then reprobed with a randomly
primed probe to human GAPDH.
|
|
 |
DISCUSSION
|
---|
We have described the cloning, expression, and characterization of
a 3
-HSD obtained from a human prostate cDNA library. This cDNA clone
appears to be virtually identical to type 2 3
-HSD originally cloned
from human liver (27). The recombinant protein was purified to
homogeneity from E. coli and kinetically characterized. Our
study is the first documentation of the properties of a 3
-HSD
expressed in human prostate and the first detailed study of the
properties of homogenous type 2 3
-HSD. Kinetic analysis demonstrates
that this protein is more accurately described as a 3
-/17ß-HSD.
The distribution of AKR-related 3
-HSD mRNA across human tissues and
within prostate cell types is described. We have shown that type 2
3
-HSD will inactivate 5
-DHT by forming 3
-androstanediol
but fails to work as a molecular switch by converting
3
-androstanediol to 5
-DHT.
Our cloning indicates that the human prostate contains multiple members
of the AKR superfamily. HSDs of this superfamily can be isoforms of
3
-, 17ß-, or 20
-HSDs. Khanna et al. (27) cloned type
1 and 2 3
-HSDs from human liver and showed that type 1 3
-HSD was
present only in the liver and is identical in sequence to DD4 (37) and
chlordecone reductase (38). It was subsequently shown that type 1
3
-HSD was not found in the prostate (39). Type 2 3
-HSD had a
broader expression pattern and was detected in the brain, kidney,
liver, lung, placenta, and testis (27). The presence of type 2 3
-HSD
in prostate was not documented. Our tissue-specific Northern analysis
confirms the previously reported distribution of 3
-HSD mRNA and
indicates that transcripts similar in size and sequence to type 2
3
-HSD are found in the prostate, blood leukocytes, colon, small
intestine, ovary, thymus, spleen, pancreas, skeletal muscle, and heart.
The Northern analysis shown here is not specific for type 2 3
-HSD
and probably detects all the 3
-HSD isoforms expressed. The high mRNA
levels observed in liver, kidney, and intestine are consistent with the
known function of 3
-HSD in these tissues where the different
isoforms have roles in bile acid biosynthesis (liver) and xenobiotic
metabolism (liver, kidney, and small intestine) (24, 36). Recently,
another cDNA clone designated type 3 3
-HSD (28), which is nearly
identical to human liver bile acid-binding protein/DD2 (35, 36, 37), has
been isolated from a human prostate cDNA library and shown to display
3
-HSD activity when transiently expressed in human embryonal kidney
(293) cells.
By overexpressing and purifying recombinant type 2 3
-HSD, we have
shown that the protein is similar to the 3
-HSDs partially purified
from rat and human prostate (16, 17, 23). Although previously reported
kinetic parameters for prostatic 3
-HSD activity were determined with
crude tissue homogenates, cellular fractions, or partially purified
enzymes (16, 18, 20, 21, 22), our characterization yielded comparable
results with these earlier studies. For example, Taurog et
al. (16) reported Km values for 5
-DHT and
3
-androstanediol in the low micromolar range with the partially
purified rat prostatic enzyme, and it preferentially used NADPH over
NADH as cofactor. The ability of human type 2 3
-HSD to oxidize
androsterone when expressed in E. coli sonicates has been
reported (27). In the present study, we were unable to detect this
activity using the same spectrophotometric assay in E. coli
sonicates. However, we could detect androsterone oxidation with a
radiometric assay using purified homogeneous protein. The predicted
amino acid sequence we describe here differs from the previously
reported sequence for human type 2 3
-HSD by only two residues. Based
on the three-dimensional structures of rat liver 3
-HSD (30, 31, 32), the
two substituted amino acids are not in the vicinity of the active site
or the substrate-binding pocket, they are on the periphery of the
structure and are unlikely to affect function. Therefore, the
differences in the specific activity for androsterone oxidation
catalyzed by the two recombinant proteins cannot be explained by
differences in sequence.
The directionality of 3
-HSD activity in the prostate has been a
point of debate. Studies of dog (13, 14) and rat (15) prostate suggest
that the dehydrogenase activity of 3
-HSD is favored over the
reductase activity, whereas other investigations of dog (18), rat (16, 17), and human (19) prostate indicate that the reduction of 5
-DHT is
preferred.
Based on kinetic characterization of purified recombinant type 2
3
-HSD, this protein can reduce 5
-DHT into 3
-androstanediol but
does not catalyze the reverse reaction. When 3
-androstanediol is
used as substrate, the 17ß-alcohol is oxidized preferentially over
the 3
-alcohol to generate androsterone which is converted to
androstanedione over time demonstrating that type 2 3
-HSD is also a
17ß-HSD. This dual activity is not unprecedented. Several HSDs of the
AKR superfamily demonstrate this phenomenon. For example, although
human liver DD1 is predominantly a 20
-HSD, it also has 3
-HSD
activity (40). Likewise, type 3 3
-HSD, which is virtually identical
to human bile acid-binding protein, displays mainly a 3
-HSD activity
but exhibits a slight 20
-HSD activity (28). Additionally, Labrie
et al. (41) have reported the cloning of a type 5 17ß-HSD,
which shares 99.1% sequence identity to type 2 3
-HSD. However, the
sequence of type 5 17ß-HSD has not been reported. The ability of HSDs
in the AKR superfamily to be positionally selective and stereospecific
on the one hand (e.g. rat liver 3
-HSD) and to be
promiscuous on the other (e.g. 3
-/20
- and
3
-/17ß-HSDs) raises issues of steroid pocket architecture. This is
particularly intriguing with regard to 3
-/17ß-HSD activity, since
in this instance steroid substrates would bind backward (D-ring
enters the active site first instead of the A-ring) and upside down
(ß-face of the steroid in the
-face orientation) to maintain
stereochemistry of hydride transfer.
Previous studies on the directionality of 3
-HSD activity in the
prostate have never considered the interplay of multiple 3
-HSD
isoforms with subtle differences in substrate specificity. It is
possible that differential expression of such isoforms would affect the
balance between 5
-DHT and 3
-androstanediol within the prostate.
As described above, at least two 3
-HSD isoforms have been detected
in the human prostate. While type 2 3
-HSD may not function as the
molecular switch between potent and weak androgens, it is well suited
to decreasing 5
-DHT levels in the prostate by forming
3
-androstanediol. Considering the fact that human bile acid-binding
protein/DD2 also functions as a 3
-HSD (35), type 3 3
-HSD may be a
better candidate to catalyze the formation of 5
-DHT from
3
-androstanediol. However, complete characterization of the
substrate specificity of type 3 3
-HSD for androgens has not been
performed.
The colocalization of 3
-HSD, 5
-reductase, and the AR within the
prostate may also influence androgen metabolism and androgen action. It
has been suggested that the stroma is metabolically active and mediates
the effect of androgens on the epithelium through epithelial-stromal
interactions (42, 43). Northern analysis on primary cultures of
epithelial and stromal cells derived from normal, BPH, and PCA tissues
using the cDNA 3'-UTR as a probe indicated that 3
-HSD mRNA
transcripts were found predominantly in epithelial derived cell
cultures. This distribution compares favorably with the reported
localization of 3
-HSD enzyme activity in epithelial cells (19, 44).
Immunohistochemical studies of adult human prostate tissues showed that
type 2 5
-reductase was predominantly expressed in stromal cells,
specifically in fibroblasts, with little expression in basal or luminal
epithelial cells. In contrast, type 1 5
-reductase and the AR were
found in both stromal and epithelial cells (45). The colocalization of
3
-HSD with the AR in epithelial cells (which are devoid of type 2
5
-reductase) and our kinetic studies suggests that type 2 3
-HSD
may protect the AR from binding potent androgens. The higher levels of
3
-HSD in primary cultures from diseased prostate may reflect either
a protective measure to decrease the effect of androgens on abnormal
growth of the prostate or may represent a movement toward androgen
independent growth of the gland. In this regard it will be interesting
to determine whether growth factors modulate type 2 or type 3 3
-HSD
expression.
 |
MATERIALS AND METHODS
|
---|
Materials
All prostate samples were obtained from human patients with
their informed consent, and the study was approved by the Institutional
Review Board of Stanford University. The human prostate cDNA library
and human prostatic poly(A)+ mRNA were obtained from
CLONTECH Laboratories (Palo Alto, CA). The pBluescript II ks-vector was
from Stratagene (La Jolla, CA), the pET-16b prokaryotic expression
vector was from Novagen (Madison, WI), and the pCRII PCR cloning vector
was purchased from Invitrogen (San Diego, CA). Blots containing
poly(A)+ mRNA from multiple human tissues were purchased
from CLONTECH Laboratories. Radiolabeled [4-14C]-5
-DHT
(58.3 mCi/mmol), [4-14C]testosterone (57.3 mCi/mmol),
[9,11-3H(N)]-3
-androstanediol (40.0 Ci/mmol),
[9,11-3H(N)]androsterone (57.0 Ci/mmol), and
[
32P]dATP (3,000 Ci/mmol) were obtained from
NEN/DuPont (Boston, MA). All other steroids were purchased from
Steraloids (Wilton, NH). Nucleotide cofactors were obtained from
Boehringer-Mannheim (Indianapolis, IN). All other compounds were
ACS grade or better from Sigma Chemical Co. (St. Louis, MO).
Screening of the cDNA Library and DNA Sequencing
A total of 1.2 x 106 plaque forming units were
screened from a prostate cDNA library in
gt10 obtained from a
22-yr-old normal Caucasian male. Screening was performed using random
primed [
-32P]dATP-labeled human colon DD1 (46) as the
probe. DD1 is a member of the AKR superfamily with 20
(3
)-HSD
activity (40). Positive clones were plaque purified, and a partial
sequence was obtained using an insert screening amplimer set (CLONTECH)
flanking both ends of the EcoRI insertion site of
gt10
vector. The 5'-end insert screening amplimer
(5'-AGCAAGTTCAGCCTGGTTAAG-3') was 8 bp upstream from the
EcoRI site, and the 3'-end insert screening amplimer
(5'-GGGACCTTCTTTATGAGTATT-3') was 22 bp downstream of the
EcoRI site. A clone containing a nearly full-length cDNA
with the highest nucleotide sequence identity to the human colon DD1
cDNA was excised from the
gt10 vector using EcoRI partial
digestion (an internal EcoRI site was present at 854 bp of
the cDNA) to retrieve the entire cDNA. A single 1.2-kb cDNA was
purified from the partial digests by agarose gel electrophoresis and
subcloned into the EcoRI site of pBluescript II ks-vector
for dideoxysequencing. Both strands of the cDNA were sequenced using
discrete 5'- and 3'-primers.
Since the cDNA obtained did not contain an ATG translation start
codon, the 5'-cDNA was regenerated from a PCR using the template
obtained from the 5'-RACE using the Marathon cDNA Amplification kit
(CLONTECH). Double-stranded cDNA was synthesized from human prostatic
poly(A)+ mRNA (CLONTECH) using oligo (dT)1218
primers followed by adapter ligation. The 5'-RACE was amplified from
the double-stranded cDNA using an adapter primer and a 3'-primer
complementary to +630 to +610 bp (5'-ATCTTTCGACTTGCAGAAATC-3') of the
cloned 3
-HSD cDNA and Vent DNA polymerase (New England Biolabs,
Beverly, MA). The amplified product was purified from an agarose gel
and subcloned into the pCR II vector (pCRII-5'-3
-HSD).
Expression Vector Construction, Overexpression, and Purification
of Recombinant Protein
To establish a prokaryotic expression construct, the full-length
cDNA was generated from the 5'-RACE product and the cDNA clone obtained
from library screening. The 5'-end of the cDNA amplified from
pCRII-5'-3
-HSD using a mutated 5'-primer
(5'-GTGACAGGCCATGGATTCCAA-3', -10 to +11 bp) and a
3'-primer (+630 to +610 bp) where the underline indicates
the changes used to generate an NcoI site. The PCR-generated
5'-end of the cDNA was digested with NcoI at both the 5'-end
and at an internal NcoI site at +450 bp to generate sticky
ends. The 3'-end of the cDNA was excised from the pBluescript II ks-
vector by digesting with NcoI and BamHI. The
digested 5'- and 3'-fragments were ligated into pET-16b vector
linearized with NcoI plus BamHI to generate the
complete pET16b-3
-HSD expression construct. The orientation of the
5'-fragment was confirmed by dideoxysequencing. The construct was
transformed into competent E. coli strain DE3(BL21) cells
under the control of the lacUV5 promoter. Cells were grown overnight at
37 C in the presence of 100 µg/ml ampicillin. Aliquots of the
overnight culture were transferred to 2-liter cultures and growth was
monitored spectrophotometrically until A600 nm =
0.6. Protein expression was then induced by addition of 1
mM isopropyl-ß-D-thiogalactopyranoside. After
4 h of incubation, cells were pelleted by centrifugation for lysis
by sonication. Overexpressed protein was purified from E.
coli sonicates using DE-52 cellulose anion-exchange and Blue
Sepharose affinity column chromatography with protein quantification,
SDS-PAGE, and Western blot analysis performed as previously described
(33).
Kinetic Characterization
Specific activities of pure recombinant type 2 3
-HSD were
measured using a Beckman DU-640 spectrophotometer (Beckman Instruments,
Fullerton, CA) by monitoring the change in absorbance of pyridine
nucleotide at 340 nm. Specific activities were determined in a 1-ml
assay system containing 100 mM potassium phosphate (pH 6.0
or 7.0) or 50 mM glycine/NaOH (pH 9.0) with 4%
acetonitrile as cosolvent at 25 C. Cofactor concentrations were 2.3
mM for NAD+ and NADP+ and 200
µM for NADH and NADPH. Substrate concentrations were 75
µM androsterone; 35 µM 5
-DHT,
3
-androstanediol, and androstanedione; and 50 µM
estrone, 17ß-estradiol, 4-pregnene-3,20-dione, and
4-pregnen-20
-ol-3-one. The fluorescence titration method used to
determine the Kd for NADPH has been described elsewhere
(33).
Vmax, kcat, and Km values for
reduction of the following substrates were obtained
spectrophotometrically using the above assay system at pH 7.0 by
varying the substrate concentration as indicated and by maintaining a
constant NADPH concentration (200 µM): 5
-DHT
(3.552.5 µM), androstanedione (1.8556.25
µM), 9,10-phenanthrenequinone (5.0200
µM), and 4-nitrobenzaldehyde (0.0501.5 mM).
Measurements of 3
-androstanediol (1.7535 µM)
oxidation were performed spectrophotometrically using the same assay
system with 2.3 mM NADP+. Kinetic constants
were calculated using the ENZFITTER (BioSoft, Cambridge, U.K.)
nonlinear regression analysis program (47) to fit untransformed data
with a hyperbolic function, as originally described by Wilkinson (48),
yielding estimated values and their associated SEs.
For increased sensitivity, a fluorometric assay was used to determine
the kinetic constants for oxidation of 3
-androstanediol. This assay
monitors the fluorescence emission of NADPH at 450 nm (slit width 10
nm) with excitation at 340 nm (slit width 5 nm) on a Perkin-Elmer
(Norwalk, CT) model 65010 M fluorometer at 25 C. Each
cuvette contained a 1-ml reaction mixture consisting of 100
mM potassium phosphate, pH 7.0, 2.3 mM
NADP+, and 3
-androstanediol (2.040 µM)
with 4% acetonitrile as cosolvent. The data were analyzed as
above.
A radiochemical assay was used to determine the kinetic constants for
the reduction of 5
-DHT. This assay provides increased sensitivity
and enabled product identification. Assay systems contained 100
mM potassium phosphate buffer, pH 7.0, 2.3 mM
NADPH, and 40,000 cpm of [14C]-5
-DHT in a 100 µl
reaction volume. Vmax, kcat, and Km
values for 5
-DHT reduction were obtained by varying the steroid
concentration (3.838.2 µM). All reactions were
incubated for 30 min at 37 C with aliquots taken every 10 min to
calculate initial velocities. Over this time course, the reaction rate
was linear. Reactions were quenched by the addition of 400 µl of
ethyl acetate, and the resulting extracts were evaporated to dryness
and re-dissolved in 40 µl methanol and applied to LK6D Silica TLC
plates. Chromatograms were developed in chloroform-ethyl acetate (4:1,
vol/vol). The positions of the substrate (5
-DHT with RF
value 0.44) and product (3
-androstanediol with RF value
0.25) were detected by reference to standards and were visualized by
spraying with a 1:1 methanol/H2SO4 solution and
heating. The amounts of substrate and product were quantitated by
scrapping the corresponding sections of the TLC plate into a
toluene-based scintillation fluid, detecting
[14C]-radioactivity with a scintillation counter, and
converting the corrected counts per min into nanomoles of product using
the final specific radioactivity of [14C]-5
-DHT.
Recovery of the radioactive steroid was greater than 90% and varied by
less than 5% between samples.
Kinetic constants for the oxidation of androsterone,
3
-androstanediol, 5
-DHT, and testosterone were determined using
similar radiometric assays. Reactions contained 100,000 cpm of either
[3H]-androsterone (3.7575 µM) or
[3H]-3
-androstanediol (1.530 µM) or
40,000 cpm of either [14C]-5
-DHT (3.838.2
µM) or [14C]-testosterone (2.550
µM). All reactions were performed with 2.3 mM
NADP+. Separation and identification of products used the
same solvent system and method as above.
Tissue Distribution of 3
-HSD mRNA
Blots containing poly(A)+ RNA from multiple human
tissues were hybridized to a 176-bp DNA probe corresponding to 3
-HSD
3'-untranslated region (3'-UTR; 995-1170 bp), which was released from
pBluescript II ks- vector (AccI and XhoI double
digestion) and random primed with radiolabeled
[
-32P]-dATP with final specific activities of greater
than 109 cpm/µg DNA. The 3
-HSD 3'-UTR sequence matches
only 3
-HSD/DD sequences from GenBank and EMBL using BLAST (49).
Hybridization was performed at 42 C for 18 h, and blots were
washed with 0.1 x standard saline citrate plus 1% SDS at 60 C
for 30 min. The blots were subjected to autoradiography at -70 C. The
blots were then stripped and reprobed with a random primed human GAPDH
cDNA probe.
Northern Blot Analysis of 3
-HSD mRNA in Primary Cultures of
Human Prostate Cells
Primary cultures of stromal and epithelial cells were prepared
from small wedges of tissues dissected from radical or open
prostatectomy specimens. After inking to mark areas of tissue removal,
prostate specimens were fixed and serially sectioned, and the histology
of each area of origin was determined. Cells derived from BPH and PCA
tissue were surrounded by cells of similar histology. Tissues were
minced and digested with collagenase. Epithelial and stromal cell
cultures were established as previously described (50). Cellular RNA
was extracted from these cultures by phenol at acidic pH. Total RNA (10
µg) was separated on 1% agarose/formaldehyde gels and transferred to
Durolon-UV membranes (Stratagene). Transferred RNA was fixed to the
membrane using a UV cross-linker (Stratalinker, Stratagene) followed by
hybridization with [
-32P]-dATP random primed 3
-HSD
cDNA probe (+1 to +854 bp) or a probe corresponding to the 3
-HSD
3'-UTR (see above). Hybridization conditions and reprobing with human
GAPDH were conducted as described. After autoradiography, the results
were quantitated using a video densitometer and the UNISCAN software
(Analtech, Newark, DE).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Trevor M. Penning, Department of Pharmacology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104-6084.
This work was supported by NIH Grants DK-47015, CA-39504, and CA-55711
(to T.M.P.).
1 Both of these authors have contributed equally to this work. 
2 Abbreviations and trivial names used are:
3
-androstanediol, 5
-androstane-3
,17ß-diol; androstanedione,
5
-androstane-3,17-dione; androsterone,
5
-androstan-3
-ol-17-one; DD, dihydrodiol dehydrogenase (EC
1.3.1.20); 5
-DHT, 5
-dihydrotestosterone
(5
-androstane-17ß-diol-3-one); 3
-HSD, 3
-hydroxysteroid
dehydrogenase (EC 1.1.1.213, formerly EC 1.1.1.50 renamed due to A-face
stereospecificity); 5
-reductase, 3-oxo-5
-steroid-4-dehydrogenase
(EC 1.3.99.5); testosterone, 4-androsten-17ß-ol-3-one. 
3 The nomenclature of the aldo-keto reductase
superfamily was recommended by the 8th International Symposium on
Enzymology and Molecular Biology of Carbonyl Metabolism, held in
Deadwood, South Dakota, on June 29July 3, 1996 (25 ). 
Received for publication June 6, 1997.
Revision received August 22, 1997.
Accepted for publication September 2, 1997.
 |
REFERENCES
|
---|
-
Bruchovsky N, Wilson JD 1968 The conversion of
testosterone to 5
-androstan-17ß- ol-3-one by rat prostate in
vivo and in vitro. J Biol Chem 243:20122021[Abstract/Free Full Text]
-
Wilson JD, Gloyna RE 1970 The intranuclear metabolism of
testosterone in the accessory organs of reproduction. Recent Prog Horm
Res 26:309336[Medline]
-
Fang S, Anderson KM, Liao S 1969 Receptor proteins for
androgens: on the role of specific proteins in selective retention of
17ß-hydroxy-5
-androstan-3-one by rat ventral prostate in
vivo and in vitro. J Biol Chem 244:65846596[Abstract/Free Full Text]
-
Mainwaring WI 1969 A soluble androgen receptor in the
cytoplasm of rat prostate. J Endocrinol 45:531541[Medline]
-
Siiteri PK, Wilson JD 1970 Dihydrotestosterone in prostatic
hypertrophy, I: the formation and content of dihydrotestosterone in the
hypertrophic prostate of man. J Clin Invest 49:17371745[Medline]
-
Gloyna R, Siiteri PK, Wilson JD 1970 Dihydrotestosterone in
prostatic hypertrophy, II: the formation and content of
dihydrotestosterone in the hypertrophic canine prostate and the effect
of dihydrotestosterone on prostate growth in the dog. J Clin
Invest 49:17461753[Medline]
-
Lloyd JW, Thomas JA, Mawhinney MG 1975 Androgens and
estrogens in the plasma and prostatic tissue of normal dogs and dogs
with benign prostatic hypertrophy. Invest Urol 13:202222
-
Hammond GL 1978 Endogenous steroid levels in the human
prostate from birth to old age: a comparison of normal and diseased
tissues. J Endocrinol 78:719[Abstract]
-
Meikle AW, Collier ES, Stringham JD, Fang S, Taylor GN 1981 Elevated intranuclear dihydrotestosterone in prostatic hyperplasia of
aging dogs. J Steroid Biochem 14:331335[CrossRef][Medline]
-
Geller J, Albert J, Loza D, Geller S, Stoeltzing W, de la Vega
D 1978 Dihydrotestosterone concentrations in human prostatic cancer
tissues. J Clin Endocrinol Metab 46:440444[Medline]
-
Krieg M, Bartsch W, Janssen W, Voigt KD 1979 A comparitive
study of binding, metabolism, and endogenous levels of androgens in
normal, hyperplastic, and carcinomatous human prostate. J Steroid
Biochem 11:615624[CrossRef][Medline]
-
Liao S, Liang T, Fang S, Castaneda E, Shao TC 1973 Steroid
structure and androgenic activity: specificities involved in the
receptor binding and nuclear retention of various androgens. J
Biol Chem 248:61546162[Abstract/Free Full Text]
-
Issacs JT 1983 Changes in dihydrotestosterone metabolism and
the development of benign prostatic hyperplasia in the aging beagle. J
Steroid Biochem 18:749757[CrossRef][Medline]
-
McKercher G, Chevalier S, Roberts KD, Bleau G, Chapdelaine
A 1984 5
-Reductase and 3
-hydroxysteroid dehydrogenase
activities in isolated canine prostatic epithelial cells. J Steroid
Biochem 21:549554[CrossRef][Medline]
-
Span PN, Sweep CGJ, Benraad TJ, Smals AGH 1996 3
-Hydroxysteroid oxidoreductase activities in dihydrotestosterone
degradation and back-formation in rat prostate and epididymis. J
Steroid Biochem Mol Biol 58:319324[CrossRef][Medline]
-
Taurog JD, Moore RJ, Wilson JD 1975 Partial characterization
of the cytosol 3
-hydroxysteroid:NAD(P)+ oxidoreductase
of rat ventral prostate. Biochemistry 14:810817[Medline]
-
Inano H, Hayahi S, Tamaoki B 1977 Prostate
3
-hydroxysteroid dehydrogenase: its partial purification and
properties. J Steroid Biochem 8:4146[CrossRef][Medline]
-
Jacobi GH, Moore RJ, Wilson JD 1978 Studies on the mechanism
of 3
-androstanediol-induced growth of the dog prostate.
Endocrinology 102:17481755[Abstract]
-
Krieg M, Weisser H,Tunn S 1995 Potential activities of
androgen metabolizing enzymes in human prostate. J Steroid Biochem Mol
Biol 53:395400[CrossRef][Medline]
-
Jacobi GH, Moore RJ, Wilson JD 1977 Characterization of the
3
-hydroxysteroid dehydrogenase of dog prostate. J Steroid Biochem 8:719723[CrossRef][Medline]
-
Hudson RW 1982 Studies of cytosol 3
-hydroxysteroid
dehydrogenase of human prostatic tissue: comparison of enzyme
activities in hyperplastic malignant and normal tissues. J Steroid
Biochem 16:373377[CrossRef][Medline]
-
Hudson RW 1984 Comparison of 3
-hydroxysteroid dehydrogenase
activities in the microsomal fractions of hyperplastic malignant and
normal human prostatic tissues. J Steroid Biochem 20:829833[CrossRef][Medline]
-
Amet Y, Simon B, Quemener E, Mangin P, Floch HH, Abalain JH 1992 Partial purification of 3
- and 3ß-hydroxysteroid
dehydrogenases from human hyperplastic prostate: comparison between the
two enzymes. J Steroid Biochem Mol Biol 41:689692[CrossRef][Medline]
-
Penning TM, Pawlowski JE, Schlegel BP, Jez JM, Lin H-K,
Smith-Hoog S, Bennett MJ, Lewis M 1996 Mammalian 3
-hydroxysteroid
dehydrogenases. Steroids 61:508523[CrossRef][Medline]
-
Jez JM, Flynn TG, Penning TM 1996 A nomenclature system for
the aldo-keto reductase superfamily. In: Weiner H, Lindahl R, Crabb DW,
Flynn TG (eds) Enzymology and Molecular Biology of Carbonyl Metabolism.
Plenum Press, New York, vol 6:579589
-
Pawlowski JE, Huizinga M, Penning TM 1991 Cloning and
sequencing of the cDNA for rat liver
3
-hydroxy-steroid/dihydrodiol dehydrogenase. J Biol Chem 266:88208825[Abstract/Free Full Text]
-
Khanna M, Qin KN, Wang RW, Cheng KC 1995 Substrate
specificity, gene structure, and tissue-specific distribution of
multiple human 3
-hydroxysteroid dehydrogenases. J Biol Chem 270:2016220168[Abstract/Free Full Text]
-
Dufort I, Soucy P, Labrie F, Luu-The V 1996 Molecular cloning
of human type 3 3
-hydroxysteroid dehydrogenase that differs from
20
-hydroxysteroid dehydrogenase by seven amino acids. Biochem
Biophys Res Commun 228:474479[CrossRef][Medline]
-
Pawlowski JE, Penning TM 1994 Overexpression and
mutagenesis of the cDNA for rat liver
3
-hydroxy-steroid/dihydrodiol dehydrogenase. J Biol Chem 269:1350213510[Abstract/Free Full Text]
-
Hoog SS, Pawlowski JE, Alzari PM, Penning TM, Lewis M 1994 Three-dimensional structure of rat liver
3
-hydroxysteroid/dihydrodiol dehydrogenase: a member of
the aldo-keto reductase superfamily. Proc Natl Acad Sci USA 91:25172521[Abstract]
-
Bennett MJ, Schlegel BP, Jez JM, Penning TM, Lewis M 1996 Structure of rat liver 3
-hydroxysteroid dehydrogenase/dihydrodiol
dehydrogenase complexed with NADP+. Biochemistry 35:1070210711[CrossRef][Medline]
-
Bennett MJ, Albert RH, Jez JM, Ma H, Penning TM, Lewis M 1997 Steroid recognition and regulation of hormone action: crystal structure
of testosterone and NADP+ bound to 3
-hydroxysteroid
dehydrogenase/dihydrodiol dehydrogenase. Structure 5:799812[Medline]
-
Jez JM, Schlegel BP, Penning TM 1996 Characterization of the
substrate binding site in rat liver
3
-hydroxy-steroid/dihydrodiol dehydrogenase: the roles of
tryptophans in ligand recognition and protein fluorescence. J Biol
Chem 271:3019030198[Abstract/Free Full Text]
-
Bruchovsky N, Sadar MA, Akafura K, Goldenberg SL, Matsuoka K,
Rennie PS 1996 Characterization of 5
-reductase gene expression in
stroma and epithelium of human prostate. J Steroid Biochem Mol Biol 59:397404[CrossRef][Medline]
-
Hara A, Matsuura K, Tamada Y, Sato K, Miyabe Y, Deyashiki
Y, Ishida N 1996 Relationship of human liver dihydrodiol dehydrogenases
to hepatic bile acid binding protein and an oxidoreductase of human
colon cells. Biochem J 313:373376[Medline]
-
Stolz A, Hammond L, Lou H, Takikawa H, Ronk M, Shively JE 1993 cDNA cloning and expression of human hepatic bile-acid binding protein.
J Biol Chem 268:1044810457[Abstract/Free Full Text]
-
Deyashiki Y, Ogasawara A, Nakayama T, Nakanishi M, Miyabe Y,
Sato K, Hara A 1994 Molecular cloning of two human liver
3
-hydroxysteroid/dihydrodiol dehydrogenase isoenzymes that are
identical with chlordecone reductase and bile-acid binder. Biochem J 299:545552[Medline]
-
Winters CJ, Molowa DT, Guzelian PS 1990 Isolation and
characterization of cloned cDNAs encoding human liver chlordecone
reductase. Biochemistry 29:10801087[Medline]
-
Dufort I, Soucy P, Zhang Y, Luu-The V, Cloning and
characterization of human type 2 3
-hydroxysteroid dehy-drogenase
from human prostatic cDNA library. Program of the Fifth International
Congress on Hormones, Cancer, Quebec, Canada, 1995, p 133
(Abstract)
-
Hara A, Taniguchi H, Nakayama T, Sawada H 1990 Purification
and properties of multiple forms of dihydrodiol dehydrogenase from
human liver. J Biochem 108:250254[Abstract]
-
Labrie F, Luu-The V, Lin SX, Labrie C, Simard J, Breton R,
Belanger A 1997 The key role of 17ß-hydroxysteroid dehydrogenases in
sex steroid biology. Steroids 62:148158[CrossRef][Medline]
-
Cowan RA, Cowan SK, Grant JK, Elder HY 1977 Biochemical
investigations of separated epithelium and stroma from benign
hyperplastic prostatic tissue. J Endocrinol 74:111120[Abstract]
-
Lasnitzki I, Mizuno T 1979 Role of the mesenchyme in the
induction of the rat prostate gland by androgens in organ culture. J
Endocrinol 82:171178[Medline]
-
Orlowski J, Bird CE, Clark AF 1983 Androgen 5
-reductase and
3
-hydroxysteroid dehydrogenase activities in ventral prostate
epithelial and stromal cells from immature and mature rats. J
Endocrinol 99:131139[Abstract]
-
Levine AC, Wang JP, Ren M, Eliashvili E, Russell DW,
Kirschenbaum A 1996 Immunohistochemical localization of steroid
5
-reductase-2 in the human male reproductive-tract and adult
prostate. J Clin Endocrinol Metab 81:384389[Abstract]
-
Ciaccio PJ, Tew KD 1994 cDNA and deduced amino acid sequence
of a human colon dihydrodiol dehydrogenase. Biochim Biophys Acta 1186:129132[Medline]
-
Leatherbarrow RJ 1987 ENZFITTER: A Non-linear Regression Data
Analysis Program for the IBM PC (and true compatibles). BioSoft,
Cambridge, UK
-
Wilkinson GN 1961 Statistical estimations in enzyme kinetics.
Biochem J 80:324332[Medline]
-
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ 1990 Basic
local alignment search tool. J Mol Biol 215:403410[CrossRef][Medline]
-
Kabalin JN, Peehl DM, Stamey TA 1989 Clonal growth of human
prostatic epithelial cells is stimulated by fibroblasts. Prostate 14:251263[Medline]