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Endocrinological Oncology |
-Reductase 2 in Core Needle Biopsies from Malignant and Benign Prostatic Tissue1
Departments of Clinical Pharmacology (C.B., T.G.S., A.R.), Urology (E.B., B.J.N.), and Pathology (L.E.), University Hospital, Uppsala, Sweden; Molecular Biology, Biomedical Centre (T.U.), Uppsala, Sweden; and Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas, Southwestern Medical Center (S.A.), Dallas, Texas
Address all correspondence and requests for reprints to: Anders Rane, Department of Clinical Pharmacology, University Hospital, S-751 85 Uppsala, Sweden. E-mail: Anders.Rane{at}klinfarm.uu.se
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-reductase type 2 (5-red2) is a key mechanism in the action of
androgens in the prostate and is important in the promotion and
progression of prostate diseases. Manipulation of the turnover of
androgens is thus fundamental in the pharmacological treatment
strategy.
We have developed a sensitive solution hybridization method for
quantification of the gene expression of 5-red2 in core needle
biopsies of the prostate. The 5-red2-specific messenger RNA (mRNA)
levels were measured in 50 human prostate transrectal ultrasound-guided
core biopsies obtained from 31 outpatients (median age 72, range 57–88
yr) undergoing biopsy for diagnostic purposes. Significant differences
were observed in the gene expression of 5-red2 between cancerous and
noncancerous tissue. In the 14 biopsies judged cancerous, the median
5-red mRNA levels were 3.5 amol/ng total RNA compared with 12.0
amol/ng total RNA in the biopsies showing no cancer
(P = 0.0018). The median 5-red2 mRNA level in
noncancerous tissue was thus 3.4 times higher than in the cancerous
specimens.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Development of prostatic diseases is clearly associated with a role of
androgens. Castrated males do not develop BPH (3), nor do they develop
CAP. The metabolism of testosterone in the prostate includes
bioactivation to the more potent dihydrotestosterone (DHT) catalyzed by
steroid 5-reductase (5-red) (4). Both DHT and testosterone may be
oxidized by the 17ß-hydroxysteroid dehydrogenase (17ß-HSD) to
5-androstane-3,17-dione and androstenedione, respectively (5). DHT
is further metabolized into 5-androstane-3ß, 17ß-diol, and
5-androstane-3,17ß-diol (6) (Fig. 1
).
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-reductase exists in two forms designated types 1 and 2 (7, 8, 9).
Mutations in the 5-red2 gene, leading to a defect 5-red2 enzyme,
cause a rare form of pseudohermaphroditism. The disorder
(5-reductase2 deficiency) is manifested by a feminization of male
external genitalia and a rudimentary prostate (4).
The 5-red2 isozyme is prostate specific and has an acidic pH optimum
(8, 9), whereas the 5-red1 isozyme has a neutral-basic pH optimum
and is expressed primarily in the skin and liver and only at relatively
low levels in the prostate (7). Furthermore, the two 5-red isoforms
have different inhibition profiles, leading to the development of
relatively specific inhibitors of the prostatic 5-red2 (9, 10),
which is the target enzyme in treatment of BPH (11, 12). Its role in
the development of CAP is currently being investigated in a large trial
on cancer prevention by the United States National Prostate Cancer
Chemoprevention Trial (ongoing) (12).
The 5-red2 enzyme is located mainly in the stromal fraction of the
prostate (13). DHT synthesized in the stroma by 5-red2 serves a
paracrine role in the induction, proliferation, and differentiation of
the adjacent prostatic epithelial cells (14).
The conversion of testosterone to DHT is a key mechanism in the action of androgens in the prostate and is important in the promotion and progression of prostatic disease. Therefore, it is of great interest to study the converting enzyme in the prostate.
We have developed a sensitive method for quantitation of the gene
expression of 5-red2 in core needle biopsies of the prostate. The
level and variation of the 5-red2-specific messenger RNA (mRNA) were
studied in groups of patients with clinical symptoms of BPH and CAP. We
report significant differences in the gene expression of this enzyme
between cancerous tissue and normal tissue.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tissue biopsies were obtained from patients attending the
out-patient clinic of the Department of Urology of the University of
Uppsala, Sweden. Patients were referred to the clinic because of lower
urinary tract symptoms and/or elevated serum prostatic-specific antigen
(s-PSA). Our project was approved by the Ethics Committee of the
university. Patients consenting to participate underwent transrectal
ultrasound (TRUS) and multiple core biopsies (needle diameter 1.2 mm,
notch length 15 mm, weight of biopsy 5–10 mg) from the prostate. One
or two biopsies were used for determination of 5-red2 mRNA level.
These biopsies were snap frozen on dry ice and subsequently kept at
-70 C until analysis. A parallel biopsy was taken for
histopathological diagnosis. By use of the TRUS technique it is
possible to visualize the trace of the needle and therefore get the
second biopsy absolutely adjacent to the first. The close spatial
configuration of these two biopsies allows us to assume that the biopsy
analyzed is histopathologically equivalent to that of the
pathologist’s report. Four to six additional diagnostic biopsies were
taken from these patients. In some cases, cancer was detected in the
additional biopsies. In these cases, the biopsies in the study were
considered noncancerous even if the patient had a cancer diagnosis.
A total of 50 biopsies were obtained from 31 patients (median age 72.2,
range 57–88 yr). Five patients of these 31 were receiving therapy for
their prostatic disease at the time of biopsy; two patients (1 CAP and
1 BPH patient) were on finasteride, two cancer patients were on
prednisolone, and one BPH patient was receiving terazosine. We excluded
three biopsies for which the histopathology was unclear or missing. The
histopathology of the residual 47 biopsies is shown in Table 1. We excluded prostatic intraepithelial neoplasia (PIN)
(n = 4) and prostatitis (n = 2) from the statistical
analysis. Included were 41 biopsies: 27 considered to be noncancer and
14 showing cancer. The cancerous biopsies contained 20–100% (median
94%) cancerous tissue as determined histopathologically in the
parallel biopsy.
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RNA extraction
Total RNA (totRNA) from small core biopsies (5–10 mg) was extracted using RNeasy total RNA kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s protocol. The totRNA was quantified spectrophotometrically at 260 nm.
PCR of 5-red2-specific gene fragment
For the construction of complementary RNA (cRNA) probes a 200 bp
fragment was amplified by PCR using 5-red2 complementary DNA (cDNA)
(2.4 kilobase) in a pBluescript SK vector (Stratagene, La Jolla, CA) as
template. Oligonucleotide primers for PCR amplification, which covered
codons 591–790 of the 5-red2 cDNA were constructed and designed to
include an EcoRV restriction site in the 5' end of the
forward primer, and a SacI restriction site in the 5' end of
the reverse primer: 5'-GAT ATC TGT TTC TGG AGC CAA TTT CCT (forward)
and 5'-GAG CTC AAA AGA TGA ATG GAA TAA GGG (reverse). PCR was carried
out with 100 ng DNA in 100 µL buffer, 200 mM
deoxynucleotide triphosphates, 2 U Pwo DNA polymerase (Boehringer
Mannheim, Mannheim, Germany), and 200 nM of each primer.
Temperatures were 94 C for 2 min, followed by 25 cycles of 94 C, 42 C,
and 72 C each for 1 min. The predicted size of the PCR product was
verified on a 3% agarose gel in TBE-buffer after ethidium bromide
staining.
Subcloning of PCR product
The PCR product was purified using QIAquick-spin PCR
purification kit (Qiagen GmbH), phosphorylated with T4 polynucleotide
kinase (Promega Corp., Madison, WI), subcloned into the
EcoRV site of a pBluescript KS transcription vector
(Stratagene, La Jolla, CA) and transfected into supercompetent E.
coli XL 1-Blue cells (Stratagene). Plasmids were purified from
positive clones isolated on selective agar plates [Amp,
isopropyl-ß-D-thiogalactopyranoside, X-gal (Promega)],
using Qiagen plasmid Midi kit (Qiagen). The 5-red2-specific fragment
was excised with EcoRV and SacI and subcloned
into the EcoRV/SacI site of a pBluescript KS
plasmid. After transfection, selection, and purification as above, the
pBluescript/5-red2 construct was used for synthesis of radiolabeled
cRNA probes utilized in the hybridization process.
Solution hybridization
cRNA probes were synthezised in vitro utilizing the T7 RNA polymerase promoter and radiolabeled with [35S]uridine triphosphate (Amersham International, Buckinghamshire, UK). Synthetic mRNA for construction of standard curves was synthezised from the opposite T3 RNA polymerase promoter.
Hybridization of target RNA was carried out with 35S-labeled cRNA (20,000 cpm/incubation) in solution (15, 16). The reaction was performed at 65 C overnight in microcentrifuge vials containing 0.6 M NaCl, 20 mM Tris/HCl, pH 7.5, 4 mM EDTA, 0.1% SDS, 1 mM dithiothreitol, 25% formamide, and 0.5–1.0 µg totRNA/vial. After incubation, the samples were treated for 45 min with 1 mL solution containing 40 µg RNase A, 2 µg RNase T1 (Boehringer) and 100 µg salmon sperm DNA.
Radioactive RNA-RNA hybrids protected from RNase digestion were precipitated by addition of 100 µL 6 M trichloroacetic acid, collected on a filter (Whatman GF/C, Whatman Intl., Ltd, Maidstone, UK), and quantitated by scintillation counting.
Construction of standard curves and evaluation
Standard curves for calculation of 5-red2-specific mRNA in
the samples were generated by plotting cpm values (y-axis)
vs. synthetic mRNA concentration (x-axis). The
5-red2-specific mRNA levels were calculated from the standard curve.
Data represent duplicate samples in each experiment. The variation
between duplicates was less than \ 5%. Statistical evaluation of
the data was made using the Mann-Whitney test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-red2
gene expression by solution hybridization using 5-red2-specific
radiolabeled cRNA probe.
The yield of totRNA from the biopsies was approximately 1 µg
totRNA/mg tissue. The entire 5-red2 cDNA (2.4 kb) in a pBluescript
(SK-) vector served as template in the PCR reaction for the generation
of a 200-bp fragment corresponding to codons 591–790 of the 5-red
gene (see Materials and Methods). After subcloning, the
construct was used for in vitro transcription of a cRNA
probe to be used in the hybridization process. The limit of detection
was 200–500 pg synthetic mRNA in the standard curves.
Core needle biopsies from 14 patients clinically diagnosed with BPH and
17 patients with CAP were included in the study. The clinical features
of the patients are summarized in Table 2.
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-red2-specific mRNA levels varied from 0–34 amol mRNA/ng
totRNA in the 47 biopsies analyzed (Fig. 2). The limit
of detection by the solution hybridization method was 0.5 amol mRNA/ng
totRNA. In 7 out of 47 samples 5-red2 mRNA levels were undetectable
by this method; they are referred to as 0 amol/ng totRNA in Table 1
and
Fig. 2. From 16 patients we obtained duplicate biopsies, with identical
histopathology in 13 of the 16 biopsies. The mean difference between
duplicate biopsies with identical histopathology was 5.1 amol/ng totRNA
(SD \ 4.0). None of the biopsies obtained from the five
patients receiving therapy for their prostatic disease were considered
extremes with respect to 5a-red2 mRNA expression.
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-red2
mRNA levels (noncancer and cancer) depending on the histopathological
evaluation of each sample analyzed. There were 14 cancerous biopsies
from CAP patients, 18 noncancerous biopsies from BPH patients, and 9
noncancerous biopsies from patients diagnosed with CAP (Table 1). In
addition there were four biopsies showing PIN and two biopsies showing
prostatitis. Significant differences in 5-red2 mRNA levels between
the cancerous and noncancerous groups were observed, as seen in Table 1
and Fig. 3. In the 14 biopsies judged to be cancerous,
the median 5-red2 mRNA levels were 3.5 amol/ng totRNA compared with
12.0 amol/ng totRNA in the 27 biopsies showing no cancer
(P = 0.0018). The median 5-red2 mRNA level in
noncancerous tissue was thus 3.4 times higher than in cancerous
specimens (Fig. 3).
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It is of special interest to note that biopsies showing no cancer but
which were taken from patients with prostatic carcinoma elsewhere in
their prostate (n = 9) had a median concentration of 11.5 amol/ng
totRNA, i.e. similar to the levels found in noncancerous
biopsies (Table 1). This would indicate that lower concentrations of
5-red2 mRNA found in cancerous biopsies are caused by the
histopathology of cancer in the analyzed cells, rather than a general
low value for the prostate harboring cancer.
In addition, we analyzed the material for prostatic size, age of the
patient, and serum PSA levels in relation to 5-red2 mRNA levels
(data not shown). We were unable to find any correlation between the
level of 5-red2 mRNA and any of those parameters. The study was,
however, not designed to detect differences in this respect, because
almost all patients were referred to the clinic because of elevated
s-PSA levels. Therefore, a comparison between s-PSA and 5-red2 mRNA
was precluded because of risk of bias.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-red2 enzyme in core biopsies of the
prostate. To our knowledge, this is the first quantitative study of
5-red2 mRNA in a large, well-defined, clinical patient material.
This new biochemical parameter has not been validated before in
relation to the histopathology of the prostate. The method presented is
based on solution hybridization and found to be sensitive enough to
quantitate levels of 5-red2-specific mRNA in small core biopsies of
5–10 mg each.
There was a significant difference in the 5-red2-specific mRNA
levels between cancerous and noncancerous tissue. This finding is
consistent with a recently published study by Elo et al.
(17), in which they reported a significantly higher expression of
5-red2 mRNA in BPH tissue compared with carcinoma specimens as
monitored by Northern blotting. They did not, however, quantitate the
specific 5-red2 mRNA levels in their study. The lower 5-red2 mRNA
level in cancerous tissue could be because of the relative lack of
stromal cells. This is further supported by the findings that
noncancerous tissue from cancer patients had normal 5-red2 mRNA
levels.
The coefficient of variation of the assay was 5%. As expected (Fig. 2), a larger variation was observed in the two biopsies obtained from
the same prostate. This probably reflects the heterogeneity of the
glandular histopathology. In spite of this variation, the difference
between the cancerous and noncancerous groups is highly
significant.
The design of our study did not allow any comparison between the
transcript levels and the activities of the 5-red2 enzyme, because
approximately 50 mg wet weight tissue is required for the metabolic
assay in vitro. However, the present data are in agreement
with our previous results from surgical specimens from CAP and BPH
prostates with higher DHT formation rates in BPH than in CAP (abstract
presented at the 1st Congress of the European Society of Clinical
Pharmacology, Paris, 1995). A positive correlation between 5-red2
mRNA levels and enzyme activity has also been reported by Thigpen
et al. (18). It is interesting that the immunoblotting
studies of 5-red2 in CAP and BPH tissue by Thigpen and colleagues
did not reveal any difference in protein levels. However, their study
was performed on a limited number of tissue samples. Further
investigations are required to elucidate the relation between the
levels of 5-red2 transcript, protein, and the specific enzymatic
activity in different types of prostatic tissue and disease.
This study was based on the concept that androgens have a crucial
role in the development of CAP and BPH. The ultimate androgen exposure
of the prostate depends on a number of factors. In addition to the
circulating levels of testosterone and its precursors, the local
intraprostatic metabolic pathways influence the total androgen
exposure. Whereas the circulating testosterone values decrease with
age, the intraprostatic androgen and androgen receptor levels are
maintained at an advanced age. 5-red2 is only one of several enzymes
that take part in the metabolism of testosterone (Fig. 1). These
enzymes include 5-red1 and 2; 17ß-HSD1, 2, 3, and 4; 3-HSD; and
3ß-HSD1 and 2. Therefore, a more complete picture of the activities
of the enzymes involved would be of interest to study in relation to
clinical status and treatment of the patient. This is presently a
subject of investigation in our laboratory.
Increased knowledge of the intraprostatic enzymes governing androgen metabolism and action in the prostate could be of key value in understanding the pathogenesis of BPH and CAP, the lack of action of androgen deprivation, and the mechanism of hormonal escape. Furthermore, it could contribute to the understanding of drug interactions that influence androgen action in the prostate.
We believe that methods to monitor the effect of various treatments on the prostatic enzymes will be of great value in the development of novel treatment options for CAP and possibly its early diagnosis.
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Acknowledgments |
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Footnotes |
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Received January 3, 1997.
Revised March 27, 1997.
Accepted March 28, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-reductase 2 deficiency. Endocr Rev. 14:577–593.-reductases. Proc Natl Acad Sci USA. 87:3640–3644.-reductase 2 gene in male
pseudohermaphroditism. Nature. 354:159–161.[CrossRef][Medline]
-reductase J Clin Invest. 89:293–300.
-reductase inhibition by finasterid (Proscar®) in
epithelium and stroma of human benign prostatic hyperplasia. Steroids. 59:616–620.[CrossRef][Medline]
-reductase inhibitor, finasterid. J Steroid Biochem Mol Biol. 37:375–378.[CrossRef][Medline]
-reductase 2. J Urol. 152:438–442.[Medline]
-reductase isozyme expression. J Clin Invest. 92:903–910.
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