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From the Department of Medicine/Endocrinology, Weill Medical College of Cornell University, New York, New York.
| Correspondence to: Dr Yuan-Shan Zhu, Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Weill Medical College of Cornell University, 1300 York Ave, Box 149, Room F-233, New York, NY 10021 (e-mail: yuz2002{at}med.cornell.edu). |
| Received for publication September 3, 2002; accepted for publication April 17, 2003. |
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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-reductase isozymes, type 1 and 2, is the major androgen in the
prostate cells. Thus, 5-reductase(s) are critical in determining
androgen activity in the prostate. However, it is unclear in prostate tumor
cells whether 1 or 2 5-reductase isozymes are expressed and whether
they are functionally important. In the present report, we studied the
importance of 5-reductase isozymes in the androgen induction of
prostate-specific antigen (PSA) gene expression in LNCaP prostatic tumor
cells. Treatment with either testosterone or DHT in LNCaP cells produced dose-
and time-dependent increases in PSA levels in the cell media and in PSA
messenger RNA (mRNA) levels in the cells. However, testosterone-induced but
not DHT-induced PSA gene expression was significantly inhibited by
finasteride, a 5-reductase inhibitor, in a dose-dependent manner.
Furthermore, we demonstrated for the first time that both 5-reductase-1
and 5-reductase-2 mRNAs were expressed in LNCaP cells using reverse
transcriptase-polymerase chain reaction (RT-PCR) and RT-PCR Southern blot
analysis. These results suggest that both 5-reductase isozymes are
present and functionally important in prostatic tumor LNCaP cells and that DHT
is a major mediator of androgen induction of PSA gene expression in these
cells.
Key words: 5-Reductase isozymes, testosterone, prostatic tumor cells
There are 2 major natural potent androgens, testosterone and
dihydrotestosterone (DHT), in humans and mammals
(Zhu et al, 1998). Although
testosterone is a major secretory androgen of the testes, DHT is the major
intra-cellular androgen and the major androgen at the nuclear level in the
prostate cells (Anderson and Liao,
1968; Bruchovsky and Wilson,
1968). DHT is converted from testosterone by 5-reductase
isozymes (Zhu et al,
1998).
Two 5-reductase isozymes, type 1 and type 2, have been identified in
humans and animals (Russell and Wilson,
1994). 5-Reductase-2 is the major isozyme in the human
prostate, although 5-reductase-1 is also expressed
(Russell and Wilson, 1994).
5-Reductase-2 is expressed both in prostate epithelial cells and, to a
greater extent, in prostate stromal cells
(Russell and Wilson, 1994).
5-Reductase-1 is also expressed in both prostate epithelial and stromal
cells (Bruchovsky et al, 1996).
5-Reductase activity is clearly demonstrated in prostate cancer tissues
and prostatic tumor cells (Bruchovsky et
al, 1988; Smith et al,
1994; Delos et al,
1998; Negri-Cesi et al,
1998). However, the expression pattern of 5-reductase
isozymes and the importance of 5-reductase isozymes in prostate cancer
tissues and prostatic tumor cells remain to be further elucidated.
In the present study, using prostatic tumor LNCaP cells, we analyzed
5-reductase isozyme expression and evaluated the role of
5-reductases in the androgen regulation of prostate-specific antigen
(PSA) gene expression. The results show for the first time that both
5-reductase-1 and 5-reductase-2 isozymes are present in the
LNCaP cells, and the androgen induction of PSA gene expression in these cells
is mainly mediated by DHT converted from testosterone by 5-reductase
isozymes. The demonstration of the presence and the functional importance of
both 5-reductase isozymes in prostate tumor cells provides important
information in designing a strategy for prostate cancer prevention and therapy
aimed at decreasing DHT action.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Preparation of Riboprobes
A pGEM-PSA plasmid that contained the full-length complementary DNA (cDNA)
of human PSA was kindly provided by Dr Ake Lundwall (Lund University, Malmo,
Sweden). This plasmid was linearized and the riboprobe was prepared by in
vitro transcription (Zhu et al,
2001) and purified by RNA G-50 column (Roche Diagnostic Inc,
Indianapolis, Ind). A human 5-reductase-2 riboprobe was prepared by in
vitro transcription using a template that contained the full length of human
5-reductase-2 cDNA (a kind gift from Drs Luu-The and Labrie, CHUL
Research Center, Quebec, Canada) subcloned to pGEM3zf.
RNA Extraction and PSA Messenger RNA Determination
Total cellular RNA was extracted using TriPURE reagents (Roche Diagnostic)
according to the manufacturer's instructions. The concentrations of RNA were
determined by UV absorbance at 260 nm.
The levels of PSA messenger RNA (mRNA) were determined by slot-blot
hybridization or Northern blot using a specific human PSA riboprobe (specific
activity, 6 x 108 dpm/µg). Northern blot and slot-blot
hybridization were performed as previously described
(Zhu et al, 2001). Briefly,
for Northern blot analysis, total cellular RNA (20 µg) was fractionated in
formaldehyde agarose gel and transferred overnight to nitrocellulose by a
capillary blot procedure in the presence of 20x SSC (1x SSC = 0.15
M NaCl and 0.015 M sodium citrate). For slot-blot analysis, 3 µg of total
cellular RNA was denatured and blotted on a nitrocellulose membrane. The
filter was then hybridized in 1x TESS buffer (5 mM
N-trishydroxymethyl-2-aminoethanesulfonic acid, 5 mM EDTA, 0.15 M NaCl, 0.25%
sodium dodecyl sulfate [SDS], pH 7.4) containing 1 x 106
cpm/mL of PSA riboprobe at 70°C overnight under mineral oil, then washed
and exposed at -80°C with an intensifying screen. The hybridization
signals were densitometrically analyzed in a Zenith soft laser densitometer
(model SLR-2D/1D) as previously described
(Zhu et al, 2001) or by
Phosphor-Imager (Amersham Biosciences Corp, Piscataway, NJ). An
oligo(dT)18 was end-labeled with 
-32P adenosine
triphosphate by T4 polynucleotide kinase and hybridized to the same slot-blot
membrane. The levels of PSA mRNA were normalized by the oligo
(dT)18 signal and presented as the percentage of vehicle-treated
control.
Determination of PSA Levels
The PSA levels in the plasma and cell media were determined by a
microparticle enzyme immunoassay (Abbott Laboratories, Abbott Park, Ill)
according to the manufacturer's instruction. The sensitivity of the assay is
0.07 ng/mL.
Reverse Transcriptase-Polymerase Chain Reaction Southern Blot
Analysis
Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed
using the Titan 1-tube RT-PCR system (Roche Diagnostic). A pair of primers,
5'-TCCTCGGTGAGATC ATTGAAT-3',
5'-CGAAGCTTCATTGACAGTTTTCATCAGCATTGTGG-3', located in exons 4 and
5 of the human 5-reductase-2 gene, and a pair of primers,
5'-GAGGCTTATTTGAATAC GTAAC-3',
5'-TTCTGAACTTTGGATACTCTTC-3', located in exons 4 and 5 of the
human 5-reductase-1 gene, respectively, were used. The RT-PCR
conditions were 50°C for 30 minutes, then 94°C for 2 minutes, and then
35 cycles of 94°C for 30 minutes, 58°C for 30 minutes, and 68°C
for 45 minutes, and a final cycle at 68°C for 5 minutes. A 18s internal
control system obtained from Ambion Inc (Austin, Tex) was used in some RT-PCRs
with a 1:9 ratio of 18s primer to competimer. The PCR products were
fractionated in a 2% agarose gel and visualized by ethidium bromide staining.
For Southern blot analysis, after electrophoresis, the samples were denatured,
neutralized, and transferred overnight to nitrocellulose membrane by a
capillary blot procedure in the presence of 20x SSC. The membrane was
prehybridized in a solution that contained 50% formamide, 6x SSC, 1%
SDS, 0.1% Tween-20, and 100 µg/mL transfer RNA (tRNA) for 1 hour at
70°C and then hybridized in the same solution that contained a specific
human 5-reductase-2 riboprobe (specific activity, 6x
108 dpm/µg) in a concentration of 1 x 106
cpm/mL at 70°C overnight. After hybridization, the filter was washed 2
times in 1x SSC plus 0.1% SDS solution at room temperature for 30
minutes and 1 time in 0.1x SSC plus 0.1% SDS at 65°C for 30 minutes,
then air dried and exposed with an intensifying screen.
The specificity of RT-PCR product was also verified by DNA sequencing using
a Promega fmol DNA sequencing kit (Promega, Madison, Wis). Yeast tRNA and
human 5-reductase-2 and 5-reductase-1 sense transcript prepared
by in vitro transcription were used as negative and positive controls of
RT-PCR, respectively.
Statistics
The data are presented as mean ± SEM. One-way analysis of variance
following post hoc Student-Newman-Keuls test was used to determine the
difference among multiple groups. Student's t test was used for
analyzing differences between 2 groups. P < .05 was accepted as
the level of statistical significance.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The effect of DHT was also time dependent as shown in Figure 1B. The levels of PSA mRNA in the cells were not changed at 2 hours, greatly increased by 12 hours, peaked at 24 hours (approximately 787% of control), and then decreased to approximately 400% of control by 48 hours after a single DHT treatment (10-8 M). The approximately 50% decrease in the steady-state levels of PSA mRNA at 48 hours of DHT treatment in comparison to its peak levels at 24 hours of treatment is consistent with a previous demonstration that the half-life of PSA mRNA is approximately 24 hours (Wolf et al, 1992). Furthermore, hormone metabolism and autologous down-regulation of the receptor after androgen exposure may also contribute to the decline in DHT action on PSA mRNA (Quarmby et al, 1990).
A similar time-dependent change in the PSA levels in the media was also observed. The levels of PSA in the medium did not change at 2 hours (2 in control vs 2.1 ng/mL/0.3 x 106 cells in DHT) but were greatly elevated at 48 hours (28 in control vs 110 ng/mL/0.3 x 106 cells in DHT) after DHT (10-7 M) treatment. Similar dose- and time-dependent induction of PSA gene expression by testosterone was observed in LNCaP cells (data not shown). Neither DHT nor testosterone altered the size of PSA mRNA as demonstrated by Northern blot analysis (Figure 1C).
Finasteride, a 5-Reductase Inhibitor, Inhibited Testosterone
but Not DHT-Induced Increase in PSA Gene Expression in LNCaP Cells
Testosterone can be converted to DHT by the 5-reductase isozymes. To
determine whether the effect of testosterone was mediated through DHT,
finasteride, a mainly 5-reductase-2 inhibitor, was used to block the
conversion of testosterone to DHT. As shown in
Figure 2, treatment with
finasteride resulted in a dose-dependent inhibition of testosterone induction
of PSA gene expression. The testosterone-induced PSA mRNA (241 ± 26% vs
100 ± 3.8% in control, P < .01, Student-Newman-Keuls test)
in the LNCaP cells was completely inhibited by concomitant administration of
finasteride at doses of 1 µM (135 ± 12% vs 100 ± 3.8% in
control, P > .05) and 10 µM (67 ± 2.3% vs 100 ±
3.8% in control, P > .05) as shown in
Figure 2A. The testosterone
induction of PSA levels (444.5 ± 65% vs 100 ± 2.1% in control,
P < .001, Student-Newman-Keuls test) in the media was also
completely inhibited by finasteride at 10 µM (171.5 ± 22% vs 100.0
± 2.2% in control, P > .05) as shown in
Figure 2B. However, the same
finasteride treatment did not affect the DHT-induced PSA gene expression.
These data indicate that testosterone-induced PSA gene expression is mainly
mediated by conversion to DHT in LNCaP cells.
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Both 5-Reductase-1 and 5-Reductase-2 Isozymes Are
Expressed in the LNCaP Cells
Using RT-PCR analysis, the expression of both 5-reductase-1 and
5-reductase-2 mRNA in the LNCaP cells was demonstrated
(Figure 3). The demonstration
of 5-reductase-1 gene expression in LNCaP cells
(Figure 3, top panel) is
consistent with a previous report
(Negri-Cesi et al, 1998). The
presence of 5-reductase-2 mRNA in the LNCaP cells was demonstrated by
RT-PCR (Figure 3, middle
panel), as well as by RT-PCR Southern blot analysis
(Figure 3, bottom panel).
Specific 5-reductase-2 mRNA products were also detected in LNCaP cells
by RT-PCR amplification using multiple pairs of primers located in exons 1, 2,
3, and 5 of human 5-reductase-2 gene (data not shown). These data
indicate that both 5-reductase-1 and 5-reductase-2 isozymes are
expressed in prostatic tumor LNCaP cells.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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It has previously been reported that the expression of the PSA gene in LNCaP cells is regulated by androgens (Henttu et al, 1992; Montgomery et al, 1992; Lee et al, 1995; Lin et al, 2000). Multiple functional androgen response elements have been identified in the promoter region of human PSA gene, conferring androgen inducibility (Riegman et al, 1991; Cleutjens et al, 1996; Schuur et al, 1996). Due to its tissue specificity and androgen inducibility, the PSA gene has been used as a model system to study androgen action in prostate gene expression and prostate cancer.
In the studies described, both testosterone and DHT induced PSA gene
expression in LNCaP cells. However, for the first time, we showed that
testosterone-induced PSA gene expression, at both mRNA and protein levels, was
blocked in a dose-dependent manner by the 5-reductase inhibitor,
finasteride, whereas finasteride had no effect on DHT-induced PSA gene
expression (Figure 2) in these
cells. Finasteride is an effective 5-reductase inhibitor and
significantly blocks the conversion of testosterone to DHT both in in vitro
and in vivo studies (DeSchepper et al,
1991; Russell and Wilson,
1994; Steiner,
2001). These data suggest that the effect of testosterone on the
regulation of PSA gene expression in LNCaP cells was mediated via conversion
to DHT.
DHT is converted from testosterone by 5-reductase isozymes
(Russell and Wilson, 1994; Zhu et al, 1998). Two
5-reductase isozymes, type 1 and type 2, have been identified in humans
and animals. In the human prostate, although both 5-reductase isozymes
are present in epithelial cells and stromal cells, 5-reductase-2 is the
predominant isozyme mainly expressed in stromal cells
(Thigpen et al, 1993;
Russell and Wilson, 1994;
Silver et al, 1994; Bruchovsky et al, 1996). Both
5-reductase-1 and 5-reductase-2 isozymes are expressed in benign
prostate hyperplasia and prostate cancer tissues
(Smith et al, 1994).
5-Reductase isozymes are also expressed in prostate tumor cells,
including LNCaP cells (Figure
3) (Guillemette et al,
1996; Delos et al,
1998; Negri-Cesi et al,
1998). Negri-Cesi et al
(1998) have shown that
5-reductase-1 mRNA was present in LNCaP cells, but the
5-reductase-2 mRNA was undetectable, even by RTPCR. In the present
study, we confirmed the presence of 5-reductase-1 mRNA in the LNCaP
cells and detected the presence of 5-reductase-2 mRNA in LNCaP cells by
RT-PCR and RT-PCR Southern blot analysis
(Figure 3). The reason for the
discrepancy between the present results and a previous report
(Negri-Cesi et al, 1998) is
unknown. Cell culture conditions (Delos et
al, 1998) and optimization of the RT-PCR amplification are factors
to be considered.
Both 5-reductase isozymes could be functionally important in
mediating androgen actions in LNCaP cells. In the present study, we showed
that finasteride, a specific 5-reductase inhibitor with a higher
affinity to the type 2 isozyme than the type 1 isozyme
(Russell and Wilson, 1994),
inhibited testosterone-induced PSA gene expression in a dose-dependent manner.
A relatively higher concentration (approximately 10 µM) was needed to
completely block the testosterone effect
(Figure 2). Similarly,
Sutkowski et al (1996)
reported that LY300502, a specific 5-reductase inhibitor with a higher
affinity to the type 1 isozyme, also inhibited testosterone-induced cell
proliferation and PSA gene expression in a dose-dependent manner in LNCaP
cells. A higher dose (at least 1 µM) of LY300502 was also needed to
completely block testosterone action. These results provide further evidence
to support the concept that both type 1 and type 2 5-reductase isozymes
present in LNCaP cells can convert testosterone to DHT, which mediates
testosterone action. Thus, administration of high doses of finasteride,
administration of high doses of a 5-reductase-1 inhibitor, or a
combination of low doses of finasteride and a specific 5-reductase-1
inhibitor may be necessary to completely block testosterone conversion to DHT
in prostatic tumor cells, producing a maximal effect. These studies are in
progress.
Finasteride has also been reported to possess activity other than
5-reductase inhibition. Wang et al
(1997) have reported that
finasteride blocks androgen action by disturbing androgen receptor-DNA complex
formation. However, our data demonstrating that finasteride had no effect on
the DHT induction of PSA gene expression support the concept that the
inhibition of testosterone effects by finasteride is due to its inhibition of
5-reductase isozymes, not to its disruption of the receptor-DNA
complex. In addition, the doses (25 to 400 µM) of finasteride used to
inhibit the receptor-DNA complex formation
(Wang et al, 1997) are much
higher than the maximal dose (10 µM) of finasteride used in the present
study.
In summary, our current study demonstrates for the first time that both
type 1 and type 2 5-reductase are present in prostatic tumor LNCaP
cells. This study also provides further evidence supporting the concept that
PSA is an androgen-inducible gene and DHT is a major mediator of the androgen
induction of PSA gene expression in LNCaP cells. These data suggest that any
strategy aimed at decreasing DHT action for prostate cancer prevention and
therapy should consider the fact that both 5-reductase isozymes are
expressed and functionally important in prostate cancer
cells.
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Acknowledgments |
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-reductase-2 plasmid and Dr Lundwall (Lund University,
Malmo, Sweden) for providing the human PSA plasmid. |
Footnotes |
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