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Departments of Urologic Surgery (J.Z., T.Z.T., S.K., R.J.M.) and Cell Biology (J.Z., S.K., R.J.M.), The Vanderbilt Prostate Cancer Center (J.Z., T.Z.T., S.K., R.J.M.), Center for Reproductive Biology Research (R.J.M.), and The Vanderbilt-Ingram Cancer Center (R.J.M.), Nashville, Tennessee 37232
Address all correspondence and requests for reprints to: Dr. Robert J. Matusik, Department of Urologic Surgery, A-1302 Medical Center North, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2765. E-mail: robert.matusik{at}mcmail.vanderbilt.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Androgens regulate the developmental expression of PB gene expression such that PB mRNA levels rise significantly in the prostatic lobes in parallel with increasing serum levels of androgens during the onset of sexual maturity (5, 10, 11). Maximal levels of PB are maintained by androgens in the adult, and castration studies have demonstrated that within 7 days postcastration, PB mRNA levels decrease as the gland regresses (11). In the castrated rat, PB mRNA levels rebound to intact levels between 12–15 days postcastration, suggesting that other factors may also regulate PB gene expression (11).
In previous transient transfection and deoxyribonuclease I footprinting studies, two distinct AR-binding sites, ARBS-1 (located at position -236 to -223) and ARBS-2 (at position -140 to -117), were identified as being required for maximal androgen induction of chloramphenicol acetyltransferase (CAT) gene expression in the human PC-3 prostate cell line (6). Neither binding site alone could induce CAT gene expression in response to androgen treatment. Also, androgen-specific transcriptional activation could not be reconstituted by linking two or three copies of either ARBS-1 or ARBS-2 alone (12). A single point mutation in either ARBS prevented the AR from binding to both sites and eliminated androgen-induced biological activity (13). Therefore, PB was androgen regulated when both AR binding sites were present in the wild-type (wt) configuration.
Androgen-selective activity was further characterized by comparing the relative binding affinities (Kd) of a purified synthetic peptide for the AR (AR2) or a synthetic peptide for the glucocorticoid receptor (GR; GR-DBD) on oligonucleotides for the consensus glucocorticoid response element (GRE) and the PB ARBS-1 or ARBS-2. As indicated in the band shift assays, the DNA binding affinity of the GR for either ARBS-1 or ARBS-2 was so low that it could not be determined, whereas the AR bound with high affinities for ARBS-1 and ARBS-2 (12). Furthermore, the substitution of the C(3)1 gene GRE with the PB ARBS-2 element converted glucocorticoid regulation of C(3)1 gene transcription into an androgen-specific response (14). As well, the PB ARBS-2 DNA element only recognized AR and not the glucocorticoid, mineralocorticoid, or progesterone receptors (15). The determinant for receptor selectivity appeared to reside in the N-terminal part of the second zinc finger as well as in a four-residue C-terminal extension of the DNA-binding domain (15). In PB gene expression, AR selectively binds ARBS-1 and ARBS-2 to regulate gene transcription, and together these two androgen-binding sites constitute the androgen response region (ARR) located between -244 to -96 bp of the PB promoter (12, 13).
The androgen-specific regulation of PB gene transcription and the prostatic epithelial cell specificity of PB gene transcription makes the promoter an ideal candidate to target transgenes to the prostate. Initial transgenic experiments used a small fragment of the PB promoter (-426 to +28 bp) linked to the bacterial CAT reporter gene. Both developmental and hormonal regulation of CAT gene expression was restricted to the transgenic prostatic epithelium; however, the levels of transgene expression were modest (7). Castration studies also showed that CAT gene expression could be restored to precastration levels with androgen treatment, but that glucocorticoid treatment had no effect (7). These data suggested that this small PB promoter lacked important enhancers that regulated PB gene expression. Therefore, a second transgenic model, using a large 12-kb PB promoter fragment (LPB) linked to the CAT gene was developed, which conferred high levels of androgen-regulated transgene expression to the transgenic prostate (16). Immunohistochemical studies showed that LPB-CAT gene expression was epithelial cell specific and that transgene expression was increased dramatically over the -426/+28PB promoter construct (16). Again, castration studies demonstrated that CAT gene expression could be restored to precastration levels with androgen treatment, but that glucocorticoid treatment had no effect, indicating that no functional GRE was present within 12 kb of the PB promoter (16).
In the current study we have developed a composite ARR2PB promoter that not only maintains prostate-specific transgene expression, but also provides a small promoter that reliably expresses high to very high transgene expression in transgenic mice. Indeed, the ARR2PB promoter resulted in high levels of transgene expression in 80% of the founders. This new construct offers a further advantage over the other PB promoter constructs in that it now exhibits glucocorticoid inducibility, and its small size makes it ideal to regulate any transgene even in viral vectors where size becomes a limiting problem.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell lines, media, and transient transfection assays
The human prostate carcinoma cell lines PC-3, LNCaP, and DU145;
the monkey kidney cell line COS-1; the human breast carcinoma cell line
MCF-7; the human pancreas carcinoma cell line PANC-1; and the human
mammary gland carcinoma cell line ZR-75–1 were obtained from
American Type Culture Collection (Manassas, VA). PC-3
cells were maintained in RPMI medium, 10% FBS, and 250 nM
dexamethasone, and the other cell lines were cultured as recommended by
American Type Culture Collection. For transient
transfection assays, cells were plated at an initial density of
3–5 x 105/100-mm dish or 2–3 x
104/well in 24-well tissue culture plates and
transfected by the calcium phosphate method as previously described
(6). Briefly, the cells were exposed to the DNA
precipitate for 6 h, followed by removal of the medium and
addition of steroids for 18 h. The rat AR (rAR) expression vector
and the rat glucocorticoid receptor (rGR) expression vector were used
in the transfection experiments. Both the rAR and rGR expression
vectors use the simian virus 40 promoter to direct expression of these
transgenes. The absolute level of protein expression from each vector
may vary from cell line to cell line due to the concentrations of
transcription factors that may regulate the simian virus 40 promoter
and changes in stability of the mRNA and the stability of the receptor
proteins. This can ultimately influence the level of induction of the
reporter gene. The transfection conditions were initially optimized by
cotransfecting mouse mammary tumor virus (MMTV)-CAT with increasing
concentrations of rat AR and rat GR expression vectors
(12). These experiments determined that the optimal
amounts of each steroid receptor expression vector were similar;
therefore, all cotransfection experiments were performed using an equal
concentration of plasmid (5 µg/ml), which was the optimal rAR and rGR
concentration for the maximal response of MMTV-CAT to
dihydrotestosterone (DHT) or dexamethasone (DEX) treatment,
respectively (12). The cells transfected with PBCAT
plasmids were harvested after 24 h of incubation from the
beginning of the transfection, and CAT activity was determined by the
two-phase fluor diffusion assay using
[3H]acetyl-coenzyme (5.9 Ci/mmol;
Amersham Pharmacia Biotech, Piscataway, NJ) as the
substrate (6). The transfection efficiency was determined
by cotransfecting pRL-CMV containing the Renilla luciferase
reporter gene (Promega Corp., Madison, WI). The luciferase
activity was determined using the Promega Corp. luciferase
assay system (NOREF>E1980). The values plotted represent the mean of at least
three individual samples ± SD.
Establishment of ARR2PBCAT transgenic mouse
lines
All animal studies were conducted in accordance with the
principles and procedures outlined by Vanderbilt University council on
animal care. From the experimental cell culture results, the
ARR2PB construct was chosen for further study.
The ARR2PBCAT transgenic mice were generated by
microinjection of the DNA into the male pronucleus of a fertilized
oocyte by the Transgenic Core/ES Cell Shared Resource of the
Vanderbilt-Ingram Cancer Center. Four transgenic founders, three males
and one female, were generated. Animals were housed in standard
lighting (12 h of light, 12 h of darkness) and allowed food and
water ad libitum. Transgenic animals were identified by
PCR-based screening using isolated mouse tail DNA as described
previously (16), and the lines were maintained as the
B6D2F1 mouse strain. Transgene copy number was determined by Southern
blot and DNA slot blot hybridization (17). Mouse genomic
DNA from transgenic animals containing a single copy of the transgene,
mouse epididymal retinoic acid-binding protein (RABP) promoter linked
to the CAT transgene (mE-RABPCAT), served as an internal control
(18). A 1200-bp complementary DNA fragment of the CAT gene
was used to detect the both the ARR2PBCAT and the
mE-RABPCAT (control) transgene. In addition, as a further internal
control, a 600-bp mE-RABP DNA fragment was used to detect the
endogenous RABP gene. The DNA probes were labeled with
[32P]deoxy-CTP (3000 Ci/mmol; Amersham Pharmacia Biotech) using the Rediprimer II kit (Amersham Pharmacia Biotech).
Prostate-specific and developmental expression of the
ARR2PBCAT transgene
The levels of ARR2PBCAT gene expression in
the four lines were compared in males and females ranging from 8–9
weeks of age (at least five mice per group). Tissue samples from the
anterior (AP), dorsolateral (DLP), and ventral prostate (VP) were
microdissected for the determination of CAT activity. To determine
tissue specificity, other tissues, including brain, spleen, thymus,
heart, lung, liver, kidney, testes, muscle, adrenals, epididymis, and
seminal vesicle from transgenic males, and brain, spleen, thymus,
heart, lung, liver, kidney, muscle, adrenals, uterus, ovary, and
oviduct from transgenic females, were analyzed for CAT activity.
Proteins were extracted by homogenization in buffer containing 0.1
M Tris (pH 7.8) and 0.1% Triton X-100 buffer, and protein
concentrations were determined by protein assay (Bio-Rad Laboratories, Inc., Richmond, CA). The CAT assay was performed
as previously described (6), and results were expressed as
disintegrations per min/mg protein ± SD or, in cases
of extremely high levels of CAT activity, per µg protein.
To determine the developmental pattern of ARR2PBCAT gene expression before, during, and after sexual maturation, four to seven transgenic males per time point were killed at 1–14 weeks and 36 weeks of age. The AP, DLP, and VP were microdissected, and CAT activity for the individual lobes was determined as described above.
Immunohistochemical studies
Animals from line 3699b, aged 17 weeks (n = 2) or 36 weeks
(n = 2), were killed, and the prostates were excised. The
individual prostate lobes (AP, DP, LP, and VP) were dissected and fixed
in 4% buffered formalin. After processing and embedding in paraffin,
5-µm sections were cut for immunohistochemical detection of CAT
protein.
Sections were deparaffinized, rehydrated, and placed in 0.05 M citrate buffer, pH 6.0. Antigenic sites were exposed by microwaving the sections for 30 min at 95–99 C before the removal of endogenous peroxidase activity with 3% H2O2. Nonspecific binding was blocked by incubating the sections with 1% DIG Blocking Reagent (Roche Molecular Biochemicals, Mannheim, Germany) in 100 mM Tris-HCl and 50 mM NaCl for 30 min. The sections were incubated with anti-CAT antibody (5'-3', Inc., Boulder, CO) overnight at 4 C (1:400 dilution), washed in PBS (14 mM NaCl, 0.3 mM KCl, 1 mM Na2PO4, and 0.2 mM KH2PO4, pH 7.4), and incubated with biotinylated antirabbit IgG (1:300; DAKO Corp., Carpinteria, CA) at room temperature for 2 h. The sections were washed with PBS and incubated for 1 h with streptavidin (1:50; Vectastain Elite ABC Kit, Vector Laboratories, Inc., Burlingame, CA). After additional washes with PBS and 50 mM Tris-HCl (pH 7.4), peroxidase activity was detected using 3',3'-diaminobenzidine tetrahydrochlorate (Liquid DAB Substrate-Chromagen System, DAKO Corp.). The reaction was terminated in distilled water, and the sections were counterstained with Harris hematoxylin (Surgipath, Richmond, VA), dehydrated, and permanently mounted with cytoseal XYL (Stephens Scientific, Kalamazoo, MI).
Hormonal regulation of ARR2PBCAT gene
expression in the prostate
To determine the effects of androgen ablation and hormone
treatment on ARR2PBCAT gene expression,
transgenic males were castrated at 10–11 weeks of age by the scrotal
route. Sham-operated age-matched males served as controls (n = 5).
At 14 days postcastration, five castrated males per group received one
of the following pellets sc. Steroid pellets (Innovative Research of America, Sarasota, FL) consisted of DHT (1.5 mg/pellet), DEX
(1.5 mg/pellet), flutamide (25 mg/pellet), RU486 (5 mg/pellet), or
placebo (5-mg pellet containing no hormone). After 7 days of treatment,
the animals were killed, and CAT activity was measured in protein
extracts obtained from the anterior, dorsolateral, and ventral lobes of
the prostate. The effects of long-term castration on transgene
expression in the absence of hormone treatment were determined by
measuring the levels of CAT activity in four animals per group at each
of the following time points: 14, 28, and 42 days postcastration.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initially, CAT gene expression was determined in LNCaP cells in
response to increasing concentrations of either DHT (Fig. 1A
) or DEX (Fig. 1B). As shown in Fig. 1, the optimal concentration for steroid hormone-induced CAT gene
expression was 10-8
M DHT and 10-8
M DEX for any construct tested. At these optimal steroid
conditions, the -426/+28PBCAT construct was preferentially induced by
DHT (11-fold) compared with DEX (1.8-fold) treatment, consistent with
our previous observations about the biological activity of the wt PB
promoter, which contains one copy of the ARR (6, 13). When
a second ARR DNA sequence was added upstream of the -244PB promoter
[(ARR)-244PBCAT], CAT activity increased 88-fold in the presence of
DHT, and a 285-fold increase in CAT activity was seen when a third copy
of ARR was added [(ARR)(ARR)-244PBCAT; Fig. 1A]. Surprisingly,
when LNCaP cells were transfected with (ARR)-244PBCAT plus the GR
expression vector and treated with DEX, the levels of CAT gene activity
increased dramatically to 137-fold when ARR copy number was doubled and
301-fold when ARR copy number was tripled (Fig. 1B), compared with the
1.8-fold induction seen with -426/+28PBCAT. Therefore, the addition of
one or two ARR DNA elements appears to alter biological activity in
response to glucocorticoid hormone treatment in vitro.
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). In
contrast to the 88-fold induction observed with (ARR)-244PBCAT,
addition of the small spacer in (ARR)-286PBCAT appeared to facilitate
the up-regulation of CAT gene expression, suggesting that the spacing
alignment between the ARRs in the PB promoter and/or perhaps sequences
within this region appears to be important for maximal induction of CAT
gene transcription by androgens. The addition of a third ARR to make
(ARR)(ARR)-286PBCAT did not greatly improve the already high
levels of CAT gene expression, resulting in a 214-fold rise in CAT
activity (Fig. 1A).
The (ARR)-286PBCAT and (ARR)(ARR)-286PBCAT constructs were also
tested for glucocorticoid induction by transfecting LNCaP cells with
either construct together with the GR expression vector plus DEX
treatment (Fig. 1B). The CAT activity increased to 203-fold with
(ARR)-286PBCAT and to 238-fold with (ARR)(ARR)-286PBCAT in the
presence of glucocorticoid treatment. Again, these promoters were able
to greatly increase responsiveness to glucocorticoid treatment compared
with that seen with the parent construct -426/+28PBCAT. Indeed, the
activity of (ARR)-286PBCAT induced by glucocorticoids was virtually
equivalent (203-fold) to that induced by DHT (229-fold).
As the largest rise in CAT gene expression in the LNCaP assay
occurred when one or more ARR DNA fragments were linked to the basic PB
promoter region, these constructs were further evaluated in a second
human prostatic cell line. The results obtained in the PC-3 bioassay
showed that the response to
10-8 M DHT
treatment of (ARR)-286PBCAT gene expression was 480-fold, and that of
(ARR)(ARR)-286PBCAT gene expression was 324-fold (Fig. 2A). Treatment with DEX at
10-8 M
produced a 496-fold induction, similar to that observed with DHT
treatment (Fig. 2B). When a second ARR was added upstream of the
-244PB promoter [(ARR)-244PBCAT], CAT activity increased 79-fold in
the presence of DHT; however, only a 30-fold increase in CAT activity
was seen when a third copy of ARR was added [(ARR)(ARR)-244PBCAT]
(Fig. 2A). When PC-3 cells were transfected with (ARR)-244PBCAT plus
the GR expression vector and treated with DEX, the levels of CAT gene
activity rose from 5- to 66-fold when ARR copy number was doubled and
74-fold when ARR copy number was tripled (Fig. 2B). Again, in a second
prostatic cell line a similar trend was seen, and the data suggest that
the close proximity of multiple AR-binding regions inhibits transgene
expression in the presence of androgens and glucocorticoids.
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ARR2PB promotes basal levels of
prostate-specific CAT expression in vitro
The specificity of the ARR2PB promoter to
regulate transgene expression was determined in both prostatic and
nonprostatic cell lines. The ARR2PBCAT construct
was cotransfected with the pRL-CMV vector (transfection efficiency
control; Promega Corp.) into several prostatic (PC-3,
LNCaP, and DU145) and nonprostatic (COS-1, MCF-7, PANC-1, and ZR-75–1)
cell lines. Basal levels of CAT activity were detected in all three
prostate cancer cell lines, whereas low to negligible levels of CAT
activity were detected in the nonprostatic cell lines (Fig. 3). Compared with PC-3 cells, the basal
levels of CAT activity in ZR-75–1, MCF-7, COS-1, and PANC-1 cells were
only 23%, 6.8%, 3.7%, and 1%, respectively. These results indicate
that the ARR2PB promoter maintains a high degree
of specificity for preferential expression in human prostate cancer
cell lines.
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). For comparison, the AR or GR
expression vectors were cotransfected into the nonprostatic COS-1,
MCF-7, ZR-75–1, and PANC-1 cell lines (even if previous studies had
reported that they contained steroid hormone receptors), and the cells
were treated with or without
10-8 M DHT
(Fig. 4A) or
10-8 M DEX
(Fig. 4B). Androgen-induced transcription was highest in the PC-3
cells, followed by LNCaP cells and, lastly, DU145 cells. Lower levels
of androgen-induced CAT activity were measured in MCF-7, PANC-1, and
ZR-75–1 cells, and negligible levels were detected in COS-1 cells. The
CAT gene expression in PC-3 and LNCaP cells was 2- to 5-fold higher
than that determined in the other cell lines. In contrast,
glucocorticoid treatment resulted in higher levels of
ARR2PBCAT gene expression in DU145, COS-1, MCF-7,
and PANC-1 cells, with the greatest change occurring in COS-1 cells
(Fig. 4B). The induction of CAT gene expression in PC-3, LNCaP, and
ZR-75–1 cells with DEX was similar to that seen with androgen
treatment. Therefore, the addition of a second ARR to the endogenous PB
ARR appears to impart new characteristics to the regulation of the CAT
reporter gene. Although steroid-induced transcription was highest in
PC-3 cells, ARR2PB has the ability to up-regulate
transgene transcription in nonprostatic cells in response to androgen
and glucocorticoid treatment.
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).
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). No CAT activity was detected in any of the other
tissues tested in males or females (data not shown). Although the
levels of CAT activity varied in the prostatic lobes from the
individual ARR2PBCAT founders, the CAT gene was
expressed in all male founders and all male offspring in a
prostate-specific manner. To determine the copy number of the
transgene, mouse liver genomic DNA was isolated from the founder and
the established ARR2PBCAT lines. Southern blot
and DNA dot blot analyses were performed to estimate transgenic gene
copy number. Line 3695 had 15 copies, line 3696 had 1 copy, and line
3706 had 2 copies of the ARR2PBCAT transgene
(Table 2). The founder 3699 split into 2
sublines in the F1 generation, with line 3699a
carrying 12 copies and line 3699b carrying 4 copies of
ARR2PBCAT transgene.
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). The levels of transgene
expression were not directly related to transgene copy number. For
example, line 3695, with a copy number of 15 genes, generated the
lowest levels of CAT activity. We also compared CAT gene expression in
age-matched males from the ARR2PBCAT transgenic
lines with other previously reported PB transgenic lines generated with
the -426/+28PBCAT, a line that gave the highest expression with this
construct (7), and the 12-kb LPBCAT constructs
(16). The CAT activities in the AP, DLP, and VP of line
3699a were 2435%, 1436%, and 1754%, respectively, of those measured
in the highest expressing -426/+28PBCAT line. The CAT activities in
the AP, DLP, and VP of the highest
ARR2PBCAT-expressing line 3699a were 104%, 44%,
and 43%, respectively, of those measured in the highest expressing
LPBCAT line I. This demonstrated that the ARR2PB
minimal promoter could induce significant levels of transgene
expression. Only line 3695 showed low levels of transgene expression.
This confirmed that although the composite ARR2PB
promoter and the -426/+28PB promoter are almost of the same length,
the ARR2PB promoter is greatly superior in
inducing high levels of transgene expression in a prostate-specific
manner in transgenic mice.
The ARR2PBCAT line 3699b and line 3706 showed
similar levels of CAT activity to that seen in the -426/+28PBCAT line.
In most of the transgenic lines, the highest levels of PB-directed
transgene expression occurred in the VP, and the lowest levels occurred
in the AP (Table 2). The exception was line 3699b, in which transgene
expression was highest in the DLP, similar to that observed in LPBCAT
line VI (16). These results suggest that the site of
transgene integration and copy number not only affect the rate of
transgene expression but may also influence the level of transgene
expression in the individual lobes of the prostate.
Developmental regulation of ARR2PBCAT
expression
PB gene expression is regulated developmentally by androgens, such
that accumulation of PB mRNA increases in parallel with the rise in
serum androgens and plateaus with the onset of sexual maturity. In line
3706, a moderately expressing line, at least four males were killed per
time point at weekly intervals from 1–14 weeks and at 36 weeks of age.
The prostates of the 1-week-old animals were too small to microdissect
into individual lobes; therefore, CAT gene expression was measured as
the total CAT activity per gland. In all other age groups, the lobes of
the prostate were separated and analyzed individually (Fig. 5A). As early as 1 week of age, CAT
activity in the prostate of line 3706 mice was detectable at very low
levels. Thereafter, CAT activity increased rapidly by 2- to 3-fold each
week, reaching a peak activity of 35,550 ± 3,233 dpm/min·mg
protein in the AP by the seventh week, 54,210 ± 3,348
dpm/min·mg protein in the DLP by the eighth week, and 80,770 ±
6,053 dpm/min·mg protein in the VP by the eighth week. A small
decrease in CAT activity was seen thereafter, but CAT activity was
still maintained at this high level. Therefore, the increase in
ARR2PBCAT gene expression followed the rise and
plateau in serum androgen levels that occur during sexual
maturation.
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). In line 3699b, the
levels of prostatic ARR2PBCAT gene expression
increased 3.5-fold in the DLP and 4-fold in the VP with almost no
change in CAT activity in the AP from 3–5 weeks of age. However,
before reaching sexual maturity around 6 weeks of age, CAT transgene
expression rapidly increased 10.6-fold (AP), 8-fold (DLP), and 5-fold
in the VP. Between 6–9 weeks of age, CAT activity continued to
increase by 8.8-fold in the AP, 11.5-fold in the DLP, and 28.7-fold in
the VP. Thereafter, CAT activity was maintained at very high levels to
36 weeks, the last data point tested. The patterns of transgene
expression in the AP, DLP, and VP were similar in lines 3699b and 3706,
with the CAT activity rising dramatically during puberty and then
reaching a plateau with sexual maturity.
ARR2PB targets transgene expression to
prostatic epithelial cells
In the construction of the composite ARR2PB
promoter, the sequences between -426 and -286 were removed. This
deletion did not affect prostate-specific expression in vivo
(Table 1); however, the removal of these sequences could have
influenced the prostatic epithelial cell-specific CAT expression
observed previously in the -426/+28PBCAT transgenic mice
(7). Therefore, the individual lobes of the prostate from
male mice that were 17 and 36 weeks old (line 3699b) were
microdissected, fixed in formalin, and processed for
immunohistochemical studies using an anti-CAT antibody. The prostates
showed specific positive immunoreactivity for the CAT protein only in
the epithelial cells (Fig. 6). There were
no differences in staining pattern or staining intensity for CAT
protein between the two age groups. Control sections incubated with
normal rabbit serum showed no positive immunoreactivity in either the
epithelium or the stroma. Very faint staining for CAT protein was
observed in the anterior prostate (Fig. 6A), consistent with the low
levels of CAT activity determined for this lobe in line 3699b (Table 2). Figure 6, B and C, shows positive CAT immunostaining in the dorsal
and lateral prostate lobes, respectively. Although the amount of CAT
activity was not individually determined for the dorsal and lateral
lobes, the intensity of staining would suggest that the amount of CAT
protein is greater in the lateral lobe (Fig. 6C) than in the dorsal
lobe (Fig. 6B). This observation is consistent with the expression and
staining pattern for the endogenous PB protein in the rodent prostate
(8). Figure 6D shows strong positive CAT immunostaining in
the ventral prostate. In contrast, no positive immunoreactivity for CAT
protein was detectable in the seminal vesicles (Fig. 6E) or in controls
treated with normal rabbit serum (Fig. 6F). Positive immunoreactivity
in the nucleus of some sections was observed in both control and CAT
antibody-treated sections. This background immunoreactivity was not
alleviated by different antigen retrieval techniques, new antibody
batches, or changes in the staining method. Similar nuclear staining
with this CAT antibody has been previously reported
(18).
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). By 2 weeks postcastration, the levels
of CAT activity fell to 51% in the AP, 19% in the DLP, and 24% in
the VP compared with that observed in sham-operated controls (Fig. 7).
By 4 weeks postcastration, prostatic CAT levels decreased further, but
only marginally, and by 6 weeks postcastration, CAT activity remained
at 46% in the AP, 11% in the DLP, and 17% in the VP compared
with that in the controls. Castration experiments using the
-426/+28PBCAT and LPBCAT transgenic mice revealed that CAT activity
decreased to undetectable levels by 3 weeks (8, 16). In
contrast, postcastration transgene expression in the
ARR2PB transgenic mice was maintained at
appreciable levels even after 6 weeks of androgen deprivation.
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). In sexually
mature male mice, by 2 weeks postcastration CAT activity had decreased
to 17% of that in intact control animals in the DLP and VP, whereas
CAT activity decreased to 38% in the AP compared with the levels
observed in intact control animals. By 21 days postcastration, CAT
activity had further decreased in the VP to 7% of that in control
animals, but no further changes were observed in CAT activity in the AP
and DLP. After implanting DHT pellets in mice 2 weeks postcastration,
androgen treatment restored CAT activity to precastration levels within
1 week (AP, P < 0.002; DLP, P <
0.008; VP, P < 0.004). Compared with the
placebo-treated group, treatment with the synthetic glucocorticoid DEX
increased CAT gene expression to 42%, 22%, and 11% (AP,
P < 0.01; DLP, P < 0.05; VP,
P < 0.0001) of that in the intact control animals,
suggesting that the addition of DEX supplemented any activity induced
by adrenal glucocorticoids. In the flutamide (antiandrogen)-treated
castrated mice, castration levels of CAT activity further decreased to
31%, 10%, and 6% (AP, P < 0.05; DLP,
P < 0.05; VP, P < 0.062) of that in
intact control animals. Compared with the placebo group, adrenal
androgens were inhibited by flutamide treatment. The ant-glucocorticoid
(RU486) treatment decreased CAT activity to 16%, 6%, and 4% (AP,
P < 0.01; DLP, P < 0.02; VP,
P < 0.035) of that in intact control animals. Compared
with the placebo group, the blocking of adrenal glucocorticoids further
down-regulated CAT gene expression in the prostate of castrated mice.
This study indicates that unlike the LPB or -426/+28PB promoter
constructs, the composite ARR2PB promoter is now
regulated by both androgens and glucocorticoids in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several androgen-regulated prostate-specific genes have been identified, and these could become the stepping stones for identifying other prostate-specific regulatory proteins that may be important in prostate morphogenesis. Although the presence of a functional AR is a critical event in the development and differentiation of the prostate, its presence in many tissues in both males and females indicates that it is not sufficient to explain prostate-specific gene expression. We have shown that the rat PB promoter targets the prostate of transgenic mice (7, 16). Similarly, the human prostate-specific antigen (PSA) gene is expressed in a highly prostate-specific manner in man. A large 6-kb 5'-flanking region (19) and a 14-kb genomic PSA fragment (20) target the prostate of transgenic mice, but a smaller 600-bp PSA promoter fragment is not prostate specific (21).
Transgenic animal studies have determined that bases -426 to +28 of the endogenous PB promoter are sufficient to target prostate-specific transgene expression (7). As a result, researchers have been analyzing this promoter fragment to define potential prostate-specific elements. Analysis of this promoter fragment has centered around transient transfection assays in prostatic and nonprostatic cell lines. Within the -426/+28PB promoter resides the ARR (-244/-96 bp) of the PB gene (12), which contains two AR-binding sites, ARBS-1 at -236 to -223 bp and ARBS-2 at -140 to -117 bp (7). The size of this ARR regulatory element suggests that transcription factors other than androgens may interact with the AR to contribute to prostate-specific gene expression. Analysis of the PB promoter by Patrikainen et al. revealed that the PB sequences between positions -278 to -240 bp were critical for both androgen- and prostate-specific gene regulation in cell culture, and they concluded that an important transcription factor-binding site at position -251 to -240 bp was integral for determining tissue specificity (22). Caution must be exercised in interpreting transient transfection into prostatic and nonprostatic cell lines as a measure of tissue specificity, as expression of the PB promoter in nonprostatic cells can be induced by the cotransfection of steroid receptors (23). Interpretation of data generated in vitro assumes that 1) the critical regulatory factors that confer prostate-specific gene expression are present in prostate cancer cell lines, and 2) that transformed nonprostatic cells lines have not acquired the expression of regulatory factors critical for prostate-specific gene expression. Unfortunately, neither of these assumptions can be confirmed because the critical regulatory factors that govern prostate-specific gene expression have not been identified. Furthermore, normal prostatic cells cannot be maintained in cell culture, and therefore, we have to rely upon established prostate cancer cell lines to perform these studies. Therefore, the definitive test of tissue specificity is the transgenic mouse model.
The ARR2PB promoter conferred prostate-specific transgene expression that was developmentally and hormonally regulated in transgenic mice. These results were consistent in four separate transgenic lines. Indeed, the new ARR2PB promoter was highly effective, resulting in high levels of transgene expression in four of the five established lines (80%). No CAT activity was measured in any other tissue tested, thus confirming that ARR2PB contained the elements required for prostate-specific gene expression. In the -426/+28PBCAT transgenic mice, one of three founders (33%) had relatively high levels of transgene expression (7), whereas in LPBCAT transgenic mice, two of six founders (33%) showed high levels of transgene expression (16). In vivo, the new small composite ARR2PB promoter could increase CAT activity in the prostate up to 1436–2435% greater than that measured with the highest -426/+28PBCAT-expressing transgenic line. Similarly, expression was comparable to that observed in the highest expressing LPBCAT line. Thus, the ARR2PB construct further narrows down the prostate-specific cis-acting element as residing between -286 to +28 bp of the PB promoter and indicates that the strong hormone-responsive region of the ARR2PB, as demonstrated in vitro, is also functioning in vivo.
In most transgenic animal models, the PB constructs target higher transgene expression to the ventral lobe, where the endogenous PB protein is expressed at lower levels. The dorsolateral prostate, which actually expresses the endogenous PB protein at the highest level, generally expresses lower levels of the transgene. These observations suggest that the lobe-specific DNA sequences are missing from these PB constructs, but prostate-specific sequences are distinct and included. However, the ARR2PBCAT line 3699b does show lobe-specific expression of the transgene, suggesting that the sequences required for lobe-specific expression are present in our constructs; however, the site of transgene integration into the genome may override these signals. Similarly, the levels of transgene expression, which are not consistent with transgene copy number, may be affected by the site of transgene integration through silencing. We have previously reported this silencing effect on the -426/+28PB and LPB promoters (7, 16). Furthermore, numerous transgenes in tandem may condense into euchromatin, thereby silencing transcription through interactions between the repeats (24, 25). In addition, line 3699 split at the F1 generation to establish 3699a (12 gene copies) and 3699b (4 gene copies). Subsequent studies with line 3699b suggest that this line appears to have undergone further changes in regulation since the F1 males were first analyzed, as future generations now show increased transgene expression. This may reflect DNA imprinting, which would alter transgene expression if the methylation pattern at the site of integration changed.
Transient transfection and transgenic mouse studies have previously shown that the PB promoter in the wild-type conformation confers androgen selectivity over other steroid hormones (6, 7, 16, 26, 27, 28). In this study we have characterized the probasin ARR by combining two or three probasin ARRs. Surprisingly, this combination altered steroid hormone specificity such that transgene expression was now regulated by glucocorticoids in addition to androgens in both cell culture and transgenic mice. This novel characteristic was unexpected, because a single ARR sequence confers strong androgenic regulation of transgenes (13).
An androgen response element (ARE) was previously defined as having the identical canonical sequence and activity as the MMTV GRE. Specificity for AR and GR binding was believed to result from the interactions of the AR or GR with other coactivators. Probasin ARBSs can bind the AR, but each site alone functions poorly as an ARE (12, 13). The combination of ARBS-1 and ARBS-2 forms the ARR, which now regulates androgen-induced gene transcription in a cooperative manner (12, 13). Notably, these binding sites can differentiate between the AR and GR when they are in the wt configuration (12, 13). Furthermore, ARBS-2 is the first DNA-binding element that has been shown to be selective for transcriptional regulation by the AR (6, 12, 15, 29). Recently, AREs have been redefined as low affinity AR-binding sites, and multiple AREs are now believed to function cooperatively to regulate transcription in response to androgen treatment (3, 12, 30).
DNA binding studies have shown that ARBS-1 and ARBS-2 bind the AR selectively (12) and that the -426/+28PB promoter is transcriptionally regulated by the AR with weak activity for the GR in vitro (6). Transgenic mouse studies using the -426/+28PB and LPB have also determined that no functional GRE resides within these PB promoter constructs (7, 16, 26, 27, 28). Comparison of the -426/+28PB and ARR2PB promoters in LNCaP cells in a dose-response curve to androgens and glucocorticoids confirmed that the -426/+28PB promoter is preferentially induced by androgens (11-fold) over glucocorticoids (1.8-fold), and that overall this promoter has very weak induction of transcription. In contrast, the ARR2PB promoter in LNCaP cells showed strong induction by androgens (229-fold) and near equal induction by glucocorticoids (203-fold). Clearly, the discriminating nature between AR and GR does involve precise cis-DNA elements (12, 14, 15); however, changing the arrangement of these elements alters their activity, most likely by changing the cooperative effects between receptors and other corepressors and/or coactivators. Thus, the increased sensitivity to glucocorticoids does not appear to be promoter specific, but, rather, is due to the interactions between the multiple ARRs. It is possible that the weak individual interactions of the GR for one ARR are further stabilized by having multiple copies present in tandem, thereby allowing the cooperative interaction between these enhancer regions. The strong induction seen with the ARR2PB promoter in vitro suggests that transcriptional regulation by this promoter in vivo would be distinctly different from that seen with the -426/+28PB promoter. It is important to note that the cell culture experiments use transformed prostate cells cotransfected with a GR expression vector; this would result in of thousands of copies of GR DNA per cell. This would most likely achieve higher levels of the steroid receptor in vitro than existing levels in vivo in normal prostatic cells. In vivo, castration induces prostatic apoptosis by which the VP regresses to about 1/10th, and the DLP regresses to about 1/5th of its original size, suggesting that epithelial cells from these two lobes respond differently to androgen. The remaining atrophic epithelial cells may have lost androgen-regulated cofactors that are important for regulation of the probasin promoter. Thus, the administration of DEX to castrated mice may not be able to induce the necessary androgen regulated cofactors that are required for ARR2PB transgene expression. Therefore, either or both explanations could account for the strong glucocorticoid effects observed in vitro that are not duplicated in vivo.
Previous castration studies in the -426/+28PBCAT or LPBCAT transgenic mice showed that prostatic CAT activity dropped to undetectable levels by 3 weeks postcastration (7, 16). Similarly, the current castration study using ARR2PBCAT mice demonstrates that removal of androgens decreases CAT activity, whereas treatment with DHT restores prostatic CAT activity to precastration levels. In contrast to the -426/+28PBCAT or LPBCAT transgenic mice, relatively high levels of CAT activity were still measurable in the prostate of ARR2PBCAT mice 6 weeks postcastration, suggesting that other factors could maintain levels of transgene expression. Treatment of castrated ARR2PBCAT mice with flutamide further reduced prostatic CAT activity compared to the placebo group, suggesting that flutamide could inhibit low levels of adrenal androgens. However, the adrenal androgens alone were not sufficient to explain the continued transgene expression after castration.
In vitro experiments indicated that glucocorticoids regulated transgene expression; therefore, it is possible that the continued CAT expression after castration could be due to adrenal glucocorticoids. To test this hypothesis, pellets containing either DEX or the antiglucocorticoid, RU486, were administered to castrated mice. DEX treatment increased CAT activity (P < 0.05) in the prostate of castrated mice, whereas RU486 decreased CAT activity (P < 0.05) over and above that seen with flutamide. Thus, ARR2PB gene expression can be regulated by both androgens and glucocorticoids in vivo.
Gene therapy using prostate-specific promoters to target viral vectors to prostate cancer cells has been proposed as a new treatment for prostate cancer patients that have failed androgen deprivation therapy. Even though greater than 80% of patients show a favorable response to androgen deprivation as measured by tumor regression and a decrease in serum PSA levels, the duration of response is usually 2–3 yr (31, 32). A number of potential mechanisms involving the AR have been proposed as reasons for the failure of androgen deprivation therapy (33, 34, 35, 36, 37, 38, 39, 40). These observations indicate that the progression of prostate cancer may still involve an active AR. In gene therapy constructs that use an androgen-driven promoter, such as the PB and PSA promoters, it may be possible to take advantage of this active AR in relapsed patients. However, it would be unacceptable to treat these men with androgens to induce an androgen-dependant viral vector, because androgen treatment would also drive prostate cancer growth. This presents a major problem in using an androgen-regulated prostate-specific promoter for gene therapy.
However, men who fail androgen deprivation therapy are often treated with glucocorticoids because it improves the quality of life, and a decrease in serum PSA levels has also been reported (41, 42). As glucocorticoid treatment is currently an acceptable therapy, it could potentially be used to induce transcription of an ARR2PB therapeutic gene in a prostate-specific manner for the treatment of prostate cancer. Thus, the glucocorticoid-inducible ARR2PB promoter may provide a novel strategy for regulating transgene expression in the development of gene therapy vectors for the treatment of prostate cancer.
In conclusion, the ARR2PB promoter maintains prostate, epithelial cell specificity in vivo, suggesting that the sequence from -286 to +28 bp of the endogenous PB promoter retains the DNA sequences necessary for prostate-specific transgene expression. This new ARR2PB composite promoter provides major advantages over previous constructs. First, the composite promoter is highly consistent in generating transgenic lines with high levels of transgene expression (80%) compared with the -426/+28PB and LPB promoters (33%). This is advantageous because if only a single transgenic line is generated, it is impossible to determine the effects of the site of integration on tissue specificity or, if no transgene expression occurs, to determine whether the site of integration or the actual transgene construct resulted in inactivity. Further, if the transgenic line has low levels of transgene expression, it may be difficult to obtain a prostatic phenotype. Second, the ARR2PB promoter is small enough to link to any transgene in viral vectors for the development of therapeutic gene therapy in the treatment of prostate cancer. The added glucocorticoid regulation via this composite promoter makes it a valuable tool for the treatment of prostate cancer patients that have failed androgen deprivation therapy.
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Received March 2, 2000.
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18) in the androgen receptor gene in human prostate
cancer. Biochem Biophys Res Commun 198:74–80[CrossRef][Medline]
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, anterior lobe; 
, dorsolateral lobe; 
, ventral lobe.
