A 6-kb Promoter Fragment Mimics in Transgenic Mice the Prostate-Specific and Androgen-Regulated Expression of the Endogenous Prostate-Specific Antigen Gene in Humans
Kitty B. J .M. Cleutjens,
Hetty A. G. M. van der Korput,
Conny C. Ehren-van Eekelen,
Robert A. Sikes,
Claudia Fasciana,
Leland W. Chung and
Jan Trapman
Department of Pathology (K.B.J.M.C., H.A.G.M.vdK., C.C.E.-vE.,
C.F., J.T.) Erasmus University 3000 DR Rotterdam The
Netherlands
Department of Urology (R.A.S., L.W.C.)
Molecular Urology and Therapeutics Program University of
Virginia Charlottesville, Virginia 22908
 |
ABSTRACT
|
---|
Prostate-specific antigen (PSA) is a
kallikrein-like serine protease, which is almost exclusively
synthesized in the luminal epithelial cells of the human prostate. PSA
expression is androgen regulated. Previously, we characterized in
vitro the proximal promoter, and a strong enhancer region,
approximately 4 kb upstream of the PSA gene. Both regions are needed
for high, androgen-regulated activity of the PSA promoter in LNCaP
cells. The goal of the present study is the in vivo
characterization of the PSA promoter. Three transgenic mouse lines
carrying the Escherichia coli LacZ gene, driven by the
632-bp proximal PSA promoter, and three lines with LacZ, driven by the
6-kb PSA promoter, were generated. Expression of the LacZ reporter gene
was analyzed in a large series of tissues. Transgene expression could
not be demonstrated in any of the transgenic animals carrying the
proximal PSA promoter. All three lines carrying the 6-kb PSA promoter
showed lateral prostate-specific ß-galactosidase activity. Transgene
expression was undetectable until 8 weeks after birth. Upon castration,
ß-galactosidase activity rapidly declined. It could be restored by
subsequent androgen administration. A search for mouse PSA-related
kallikrein genes expressed in the prostate led to the identification of
mGK22, which was previously demonstrated to be expressed in the
submandibular salivary gland. Therefore, the 6-kb PSA-LacZ transgene
followed the expression pattern of the PSA gene in humans, which is
almost completely prostate-specific, rather than that of mGK22 in
mice. In conclusion, the 6-kb promoter fragment appears to contain
most, if not all, information for androgen regulation and prostate
specificity of the PSA gene.
 |
INTRODUCTION
|
---|
Prostate specific antigen (PSA) is a 30- to 33-kDa glycoprotein,
which is almost exclusively produced by the luminal epithelial cells of
the human prostate. It is one of the predominant proteins secreted into
the prostatic fluid. Serum PSA is a well known marker for diagnosis and
monitoring of prostate cancer (1, 2). The PSA gene (or KLK3) is a
member of the human kallikrein gene family. Other members of the
kallikrein gene family are the hGK-1(KLK2) gene, which is also
expressed in the prostate, and the tissue kallikrein gene (KLK1), which
is mainly expressed in the pancreas and kidney (3, 4, 5, 6). The three genes
are clustered within the 60-kb kallikrein locus on chromosome 19 (7, 8). PSA expression can be regulated by androgens (9, 10, 11). Previously,
we and others characterized in vitro the 632-bp proximal
promoter (11, 12) and a strong 440 bp-enhancer region, approximately 4
kb upstream of the transcription start site of the PSA gene (13, 14).
Both regions are required for high, androgen-regulated activity of
the PSA promoter in LNCaP cells. Two functionally active androgen
receptor-binding sites (androgen response elements, or AREs) were
identified in the proximal PSA promoter, at positions -170 (ARE-I) and
-394 (ARE-II), respectively (11, 12). The upstream enhancer showed
synergistic cooperation with the proximal PSA promoter and was found to
be composed of at least three separate, but cooperating, regulatory
regions. At -4.2 kb, the presence of a functionally active,
high-affinity androgen receptor-binding site (ARE-III) was established
(14). Transient transfection of a 6-kb PSA promoter fragment,
containing both the proximal promoter and the upstream enhancer linked
to the luciferase reporter gene, to prostate and nonprostate cell lines
showed largely LNCaP prostate cell-specific activity (13, 14). The
strong tissue specificity of the endogenous PSA gene in vivo
and the 6-kb PSA promoter fragment in transient transfection
experiments makes the PSA promoter a candidate to deliver therapeutic
genes to prostate cancer cells. To explore this view, the goal of the
present study is the in vivo characterization of the PSA
promoter in transgenic mice.
In mice, the kallikrein gene family is composed of 24 members, half of
which are probably pseudogenes (15). Although structurally related to
the PSA gene, none of the mouse kallikreins can be considered as the
mouse homolog of human PSA, because of the different tissue
distribution (16). All functional mouse kallikrein genes are expressed
in the submandibular gland. Individual genes show additional expression
in pancreas, kidney, spleen, and/or testis. Mouse kallikrein expression
in the prostate has not yet been demonstrated. Two members of the
closely related rat kallikrein gene family have been found to be
expressed in both prostate and submandibular gland (17). To compare PSA
promoter specificity in transgenic mice with the promoter specificity
of endogenous mouse kallikreins, we determined which, if any, of the
mouse kallikrein genes was expressed in prostate.
 |
RESULTS
|
---|
Activity of the PSA Promoter LacZ Fusion Constructs in LNCaP
Cells
Previously, in transfection experiments, we characterized
the proximal promoter and a strong 440-bp core enhancer region,
approximately 4 kb upstream of the transcription start site of the PSA
gene (11, 12, 14). Two functionally active AREs were identified in the
proximal PSA promoter, at positions -170 (ARE-I) and -394 (ARE-II),
respectively (11, 12). In the center of the 440-bp upstream enhancer
region, a third functionally active ARE, ARE-III (-4200), could be
demonstrated (14). Although both the proximal promoter and the upstream
region contributed to maximal androgen-regulated and cell-specific
activity of the PSA promoter, the upstream enhancer was found to be
essential for high activity (12, 14). To investigate the regulatory
regions of the PSA promoter in transgenic mice, two LacZ reporter gene
constructs were designed (Fig. 1A
). In these constructs,
the LacZ gene is driven by the 632-bp proximal PSA promoter
(PSA-4-LACH) or by the 6-kb PSA promoter fragment (PSA-61-LACH). The
hormone-induced activity of the constructs was tested in transiently
transfected LNCaP cells. The PSA-4-LACH construct, cotransfected with
the human androgen receptor expression plasmid pSVARo, was 7-fold more
active in the presence of 1 nM R1881 than in its absence
(Fig. 1A
). In the absence of pSVARo, PSA-4-LACH showed a limited
androgen inducibility (1.8-fold). Under these conditions, PSA-61-LACH
activity was induced 600-fold by R1881. These results are essentially
identical to those obtained with comparable luciferase reporter gene
constructs (12, 14).

View larger version (31K):
[in this window]
[in a new window]
|
Figure 1. Structure and Activity of the PSA-LACH Constructs
Introduced in Transfected LNCaP Cells
A, Schematic representation of constructs PSA-61-LACH and PSA-4-LACH.
The 440-bp core enhancer region (-4380 to -3940) is represented by a
hatched box; ARE sequences are indicated by black
bars. The open box represents the LacZ open
reading frame; numbered black boxes indicate exons 1 and
2 of the mouse protamine gene. Positions of primers used to identify
transgenic animals are indicated below PSA-61-LACH. B,
LNCaP cells were transiently transfected with the PSA-4-LACH and
PSA-61-LACH constructs or with PSA-4-LACH plus the androgen receptor
expression plasmid as described in Materials and Methods
and Ref. 14. Incubation with the plasmid precipitate was for 4 h.
In indicated cases, cells were incubated with 1 nM R1881
for 24 h. Induction values are given at the top of
the bars.
|
|
Identification of Transgenic Mice
Both PSA-4-LACH and PSA-61-LACH were used to generate transgenic
mice. Three PSA-4-LACH and five PSA-61-LACH founder animals were
identified by PCR of tail DNA with primers PSA-s and LacZ-as (data not
shown). Transmission of the transgene to their offspring was
demonstrated for three PSA-61-LACH and all three PSA-4-LACH transgenic
lines. One PSA-61-LACH male founder did not transmit the transgene;
another PSA-61-LACH male founder was infertile. Comparison of the
hybridization signals of the transgene and the endogenous mouse
protamine-1 gene on Southern blots of KpnI-SacI
digested genomic DNA revealed the presence of 4, 2, and 38 copies of
the transgene in lines PSA-61 TG2, TG28, and TG31, respectively (Fig. 2A
, lanes 46). PSA-4 TG1, PSA-4 TG2, and PSA-4 TG6
carried approximately 150, 100, and 126 copies of the transgene (Fig. 2A
, lanes 13). Note that the endogenous mouse protamine-1 gene showed
a restriction fragment length polymorphism, resulting in 6- and/or 8-kb
hybridizing fragments (Fig. 2A
, lanes 7, 8).

View larger version (33K):
[in this window]
[in a new window]
|
Figure 2. Characterization of Transgenic Mouse Lines
A, Southern blot analysis of the
KpnI-SacI-digested genomic DNA of
PSA-4-LACH (lanes 13) and PSA-61-LACH (lanes 46) transgenic (TG)
lines. Lane 7 contains DNA of a control mouse. DNA (10 µg/lane) was
hybridized with a 175-bp mouse protamine cDNA probe (see
Materials and Methods). By comparison of the intensity
of the endogenous (see arrowheads) and transgene bands,
the number of transgene copies present in the individual transgenic
lines was determined (numbers on top of each lane). For
PSA-4-LACH transgenic animals, two different exposure times of the same
Southern blot are shown (a 4-h exposure of the transgene-hybridizing
fragment and a 40-h exposure of the endogenous mouse protamine gene).
Note that the endogenous protamine fragment is polymorphic, leading to
a hybridizing fragment of 6 or 8 kb. B, Liquid ß-galactosidase assay
of tissue extracts of 10-week old PSA-61-LACH TG28 male mice. SMG,
Submandibular gland; SLG, sublingual gland; PG, parotid gland. C,
ß-Galactosidase activity in lateral prostate lysates of PSA-4-LACH TG
1, 2, and 6 and PSA-61-LACH TG 2, 28, and 31 animals as compared with
activity in control mice. D, RT-PCR analysis of LacZ/protamine
transgene mRNA in RNA obtained from dorsal (DP), lateral (LP), ventral
(VP), and anterior prostate (AP) and SMG of PSA-61-LACH TG 28 male
mice. Experimental details are described in Materials and
Methods. The lower panel shows the result of
RT-PCR analysis of ubiquitously expressed
glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) mRNA. PCR products
were separated over a 2% agarose gel.
|
|
The 6-kb, but Not the 632-bp, PSA Promoter Directs Lateral
Prostate-Specific Transgene Expression
To determine the expression pattern of the transgene, male mice
were killed at 8 to 16 weeks of age, and ß-galactosidase activity was
measured in 26 different tissue lysates (see Materials and
Methods). Thorough analysis of all three PSA-61-LACH transgenic
mouse lines showed exclusive ß-galactosidase activity in extracts
from lateral prostate. In all other tissues, including the dorsal,
ventral, and anterior prostate lobes, LacZ expression was undetectable
(as shown in Fig. 2B
for PSA-61 TG28). ß-Galactosidase activity could
also not be detected in extracts from tissues of virgin or lactating
female transgenic mice (data not shown). ß-Galactosidase expression
could not be found in any of the tested tissues of PSA-4-LACH mice
(data not shown, and Fig. 2C
). For PSA-61-LACH, transgene activity was
independent of the number of integrations because ß-galactosidase
activity in the lateral prostate was comparable, despite the difference
in copy numbers (4, 2, and 38, respectively). The level and specificity
of transgene expression appeared independent of the integration
site.
To screen for the presence of low levels of transgene expression in the
different prostate lobes and submandibular gland, a known expression
site of mouse kallikreins, we performed RT-PCR with transgene
cDNA-specific primers, and GAPDH as a control (see Materials and
Methods). Again, transgene expression could only be detected in
the lateral prostate (lane 2, Fig. 2D
).
PSA-61-LACH Expression Is Restricted to the Luminal Epithelial
Cells of the Lateral Prostate
Whole mount ß-galactosidase staining, followed by sectioning of
the paraffin-embedded tissue, was performed to investigate the cell
type in the prostate expressing the LacZ gene. As demonstrated in Fig. 3B
, ß-galactosidase staining was restricted to the
luminal epithelial cells. Staining was concentrated at the basal site
of the cytoplasm. No staining was found in the lateral prostate from
age-matched control mice (Fig. 3A
). To further evaluate PSA-61-LACH
expression, sections of the paraffin-embedded lateral prostate of
PSA-61-LACH-positive and control mice were analyzed by in
situ hybridization using sense and antisense digoxygenin
(DIG)-labeled protamine riboprobes. Results obtained with the antisense
protamine probe revealed that transgene mRNA was localized within the
cytoplasm of the luminal epithelial cells of the lateral prostate (Fig. 4
, C and D). No hybridization signal was detected in
control mice or with a sense protamine riboprobe (Fig. 4
, A and B). The
restricted expression of the transgene to the luminal epithelial cells
is consistent with endogenous PSA expression in the human prostate
(18).

View larger version (72K):
[in this window]
[in a new window]
|
Figure 3. Transgene Expression in the Lateral Prostate
A and B, Whole mount X-gal staining, followed by neutral red
counterstaining, of 5-µm paraffin-embedded sections of lateral
prostate of a 10-week PSA-61-LACH TG 28 male (B) and lateral prostate
of a nontransgenic littermate (A) (magnification 400x). Blue X-gal
staining is shown as blue spots in the cytoplasm of
luminal epithelial cells.
|
|

View larger version (128K):
[in this window]
[in a new window]
|
Figure 4. RNA in Situ Hybridization Analysis of
Lateral Prostate Tissue Sections of a 10-Week-Old PSA-61-LACH TG 28
Male Transgenic Mouse
Five micrometer sections of paraffin-embedded tissue were incubated
with a DIG-labeled protamine RNA probe. Hybridization was visualized
with alkaline-phosphatase-conjugated anti-DIG antibody (see
Materials and Methods). C and D, Tissue sections of a
14-week-old PSA-61-LACH male transgenic mouse; B, nontransgenic
littermate (antisense probe; 400 x magnification). A, Incubation
of a transgenic mouse prostate with a sense protamine riboprobe.
|
|
Developmental and Hormonal Regulation of PSA-61-LACH Expression
PSA gene expression has been shown to be developmentally regulated
and to follow plasma testosterone levels (19). In in vitro
studies, expression of PSA mRNA and protein PSA promoter activity are
strongly androgen regulated (9, 10, 11, 12, 13, 14). To determine the pattern of the
PSA-61-LACH transgene expression during development, lysates of lateral
prostate tissues were prepared from line 28 males between 2 and 52
weeks of age. As indicated in Fig. 5A
, the dorsolateral
prostate of 2-week-old and the lateral prostate of 4-week-old mice did
not show significant ß-galactosidase activity. In contrast, sexually
mature males, ranging from 8 to 52 weeks of age, showed an almost
constant, high level of ß-galactosidase activity (
1500 relative
light units (RLU)/µg protein).

View larger version (19K):
[in this window]
[in a new window]
|
Figure 5. Developmental (A) and Androgen (B) Regulation of
PSA-61-LACH Expression
A, ß-Galactosidase activity in extracts of dorsolateral prostate of
2-week, and lateral prostate of 4-week and 8- to 52-week old
PSA-61-LACH TG 28 mice. B, Androgen regulation of ß-galactosidase
activity in lateral prostate of PSA-61-LACH TG 28 mice. Mice were
castrated at 10 weeks of age. After 4 days, part of the mice were
supplemented with DHT or vehicle. In indicated cases, mice were
supplemented once a day with DHT (5 mg/kg body weight; in 100% ethanol
mixed with 9 vol sesame oil, and injected subcutaneously). Lateral
prostate of 10-week-old PSA-61-LACH mice and nontransgenic littermates
served as control. Data shown are the average of three mice, except for
the 8- to 52-week group in Fig. 5A , which is the average of ten animals
(±SEM).
|
|
To obtain information on androgen inducibility of the 6-kb PSA promoter
in transgenic mice, sexually mature PSA-61-LACH males of line 28 were
castrated, and ß-galactosidase activity in the lateral prostate was
determined at 4 days after castration and at 4 days after castration
followed by 2 days of hormone replacement [(5 mg dihydrotestosterone
(DHT)/kg body weight)]. As demonstrated in Fig. 5B
, transgene activity
decreased dramatically after castration and returned very rapidly to
precastration levels after DHT administration. This finding strongly
indicates androgen regulation of transgene expression.
Mouse Kallikrein Expression
To investigate mouse kallikrein gene expression in the prostate,
RNA was isolated and RT-PCR was performed with primers overlapping
highly conserved regions in exon 3 (KALK-3-s) and exon 4 (KALK-4-as) of
all known mouse kallikrein genes (see GenBank data for mouse kallikrein
sequences). Thirty-four cloned, 146-bp PCR fragments were sequenced.
Thirty-two clones contained a mGK22 fragment (20), the two additional
cDNA fragments were 94% identical, and both contained novel kallikrein
sequences, with highest homology to mGK16 (91 and 92%, respectively)
(21). Previously, mGK22 was found to be expressed in both male and
female salivary glands, but absent in all other tissues tested (22).
RT-PCR with mGK22-specific primers confirmed the presence of mGK22 mRNA
in lateral prostate and submandibular gland. mGK22 was absent in
dorsal, ventral, and anterior prostate (Fig. 6
). The
expression level in submandibular gland was much higher than in lateral
prostate.

View larger version (54K):
[in this window]
[in a new window]
|
Figure 6. RT-PCR Analysis of Mouse Glandular Kallikrein 22
Expression in the Various Lobes of the Mouse Prostate and Male
Submandibular Gland
The RT-PCR products were blotted to Hybond N+ membrane and
hybridized with a random primed 32P-labeled probe specific
for the expected 634-bp cDNA fragment. RT-PCR of GAPDH mRNA in the RNA
preparations of the different tissues is shown in the
lower part. For abbreviations see legend to Fig. 2D .
|
|
 |
DISCUSSION
|
---|
Previously, we investigated the properties of the 632-bp
proximal promoter and a strong far upstream (-4 kb) 440 bp enhancer
region of the PSA gene in transfected LNCaP cells (11, 12, 14).
Although both regions contributed to androgen-regulated
activity of the promoter, the presence of the 440-bp core enhancer was
a prerequisite for high activity. A 6-kb PSA promoter fragment, which
contains both the proximal promoter and the upstream enhancer
region, was mainly active in LNCaP prostate cells. However, PSA
promoter activity was also observed in T47D mammary tumor cells
(14).
In the present study we demonstrate the in vivo prostate
specificity of the PSA promoter. We showed that the 6-kb, and not the
632-bp, PSA promoter is able to direct reporter gene activity in
transgenic mice. In three independent transgenic lines, carrying a LacZ
reporter gene under control of the 6-kb PSA promoter, hormonally and
developmentally regulated expression of the transgene was exclusively
targeted to the luminal epithelial cells of the lateral prostate, which
mimics the expression pattern of the endogenous PSA gene in the human
prostate. This strongly suggests that the 6-kb PSA promoter contains
most, if not all, information for prostate-specific activity. The
specific expression of the transgene in the mouse lateral prostate is
in agreement with the structural homology between the human prostate
and the mouse lateral prostate, and the mouse kallikrein expression in
the lateral prostate. The variable level of PSA expression in human
breast cancer (23) and the activity of the 6-kb PSA promoter in
transiently transfected T47D human mammary tumor cells (14) could not
be confirmed in normal breast tissue of female PSA-61-LACH transgenic
mice (data not shown).
Transgene expression was assessed in a liquid ß-galactosidase assay
by RT-PCR and by RNA in situ hybridization. Additionally,
X-gal staining of the different tissues was performed. X-Gal staining
of adult mouse tissues is complicated due to high endogenous
ß-galactosidase activity present in many tissues, including testis,
epididymis, vas deferens, liver, intestine, and prostate. This problem
was overcome by modification of the standard protocols (24, 25).
Incubation at elevated temperature before staining (1 h at 50 C), and a
raised pH (8.6) during the various incubation steps (see
Materials and Methods) suppressed endogenous
ß-galactosidase activity, without noticeable loss of activity of the
E. coli-derived transgene. Only in epididymis, vas deferens,
and anterior prostate could endogenous ß-galactosidase activity be
found at a long (>16 h) staining period, which precludes detection of
a low level of transgene expression in the X-gal assay (data not
shown).
ß-Galactosidase expression was undetectable in the PSA-4-LACH mice,
despite the presence of 100 or more copies of the transgene in all
three transgenic lines. Transient transfection of LNCaP cells with
PSA-4-LACH (Fig. 1
) and also PSA-4-LUC constructs (12, 14) showed low
activity of this 632-bp promoter fragment, especially in the absence of
a cotransfected androgen receptor expression plasmid. The observation
by Schaffner et al. (26), that transgenic mice carrying a
Ha-rasT24 oncogene, driven by the 632-bp proximal PSA
promoter, developed salivary gland and gastrointestinal tract tumors
seems to be in contrast to our findings for this promoter. However,
mutant Ha-ras expression was only confirmed in salivary
gland tumors, and not in gastrointestinal tumors. Furthermore, the late
onset of tumorigenesis could indicate that Ha-ras expression was a
secondary event. This might be related to PSA expression in a subset of
salivary gland tumors in humans (27). An alternative hypothesis is that
Ha-ras intron or exon sequences affect the selectivity and
level of expression of the oncogenic transgene.
The three PSA-61-LACH transgenic lines show a comparable level of
lateral prostate-specific, but copy number-independent,
ß-galactosidase expression. This could indicate that the PSA-61-LACH
transgene cassette lacks elements, such as matrix attachment regions or
locus control regions, that might determine boundaries in chromatin
structure, leading to copy number-dependent and position-independent
activity of transgenes (see Ref. 28 and references therein). The 6-kb
PSA promoter fragment contains all DNAseI-hypersenstive sites
(which indicate important regulatory regions) in the 31 Kb region
upstream of the PSA gene (see Ref. 14). However, it might lack
putative, so far unidentified regulatory sequences within the PSA gene,
or in the flanking region downstream of the PSA gene, or even
downstream of the hGK-1(KLK-2) gene, which is also prostate
specifically expressed, and which is at a distance of 12 kb in the
human genome (7, 8). Alternative explanations for copy
number-independent activity are also possible. Although difficult
to compare, the RT-PCR and X-gal staining experiments suggest that the
expression level of the transgene in PSA-61-LACH mice is not as high as
that of the endogenous PSA gene in the human prostate. Although this
might be due to the integration site and the properties of the LacZ and
protamine part of the transgene cassette, it is a real possibility that
one or more trans-acting factors that direct high level PSA
expression in the human prostate are absent, or present in a much lower
concentration in the mouse prostate. If this is indeed the case, these
factors could limit expression of the transgene, which corresponds to a
comparable activity of the 6-kb PSA promoter in the three independent
transgenic lines. This might also explain the low expression level of
the mouse kallikrein mGK22 in the prostate. On the other hand, the
latter might be caused by differences in promoter make up. Further
analysis of mGK22 mouse kallikrein promoter activity in human prostate
cell lines should provide additional information. In this regard, it is
also interesting that the 6-kb PSA promoter-driven transgene expression
pattern was different from that of mGK22, which is expressed at a high
level in submandibular glands. The PSA-61-LACH transgene follows the
expression pattern of the endogenous PSA gene in humans, and not that
of mouse kallikreins.
The 6-kb PSA promoter is the first human promoter that directs
prostate-specific expression in transgenic mice. Previously, three rat
promoters, rKLK8, C3(1), and probasin, have been studied with respect
to prostate specificity and applicability in the development of rodent
prostate cancer models (29, 30, 31, 32, 33, 34, 35). Transgenic rats carrying a 2.5-kb
rKLK8 rat kallikrein promoter fragment did not show tissue specificity.
Expression of the transgene was demonstrated in almost all tissues
tested, including prostate, but was absent at the major sites of
endogenous gene expression, the submandibular and sublingual salivary
glands (29). Transgenic mice carrying a 6-kb 5'-flanking region of the
rat C3(1) gene linked to the ß-galactosidase reporter gene (30) or a
9.5-kb fragment carrying the C3(1) gene with 4-kb upstream and 2-kb
downstream flanking sequences (31) did not direct transgene activity
strictly to the prostate. Depending on integration site, expression was
also detected in testis, heart, lung, and skeletal muscle. Transgenic
mice bearing a 5.7-kb C3(1) promoter linked to the SV40 large T antigen
region developed at 7 months a prostate adenoma or adenocarcinoma (32).
Female mice carrying this transgene acquired mammary adenocarcinomas.
The mice also developed other phenotypic changes including several
proliferative lesions and malignancies leading to premature death.
Greenberg et al. (33) reported a 426-bp promoter fragment of
the rat probasin gene directing chloramphenicol acetyltransferase (CAT)
reporter gene expression to the prostate of transgenic mice. These
transgenic mice showed CAT expression in dorsal, lateral, and ventral
prostate. Low levels of transgene expression were observed in the
anterior prostate and in the seminal vesicles. Although prostate
specific, the expression level of the transgene was dependent on the
integration site and did not strictly follow the expression pattern of
the endogenous rat probasin gene, which is selectively expressed in the
dorsolateral prostate. Cointegration of chicken lysozyme matrix
attachment regions resulted in transgene expression in dorsolateral
prostate of adult mice. Cointegration of matrix attachment sites was
insufficient to facilitate high-level and copy number-dependent
expression. Transgenic mice carrying the 426-bp probasin
promoter-driven SV40 large T antigen oncoprotein developed progressive
forms of prostatic cancer (34, 35).
Progress toward the understanding of the biology of prostate cancer
benefits enormously from the availability of proper animal models
displaying the whole range of clinical stages. The present study
provides a baseline for the generation of such models, utilizing the
6-kb PSA promoter hooked to the appropriate oncogenes. Because of its
tissue specificity and integration site-independent, constant activity
it might even be preferred above the probasin and C3(1) promoter-driven
prostate cancer models.
The observations presented in this study are not only relevant to the
generation of mouse prostate cancer models, but also to gene therapy
programs of human prostate cancer. The PSA gene is not only expressed
in the luminal epithelial cells of the normal human prostate, but also
in almost all prostate cancers. Therefore, the regulatory elements that
determine PSA expression in prostate cancer are of potential interest
for building a promoter to drive expression of therapeutic genes in
prostate cancer cells. The strict prostate specificity of the 6-kb PSA
promoter fragment strongly supports the applicability of this large
promoter fragment, or derivatives, in gene therapy of human prostate
cancer. Preliminary experiments, indicating prostate specificity of the
6-kb promoter-driven TK gene in an adenovirus construct, are in
accordance with this view (A. Gotoh, A. S. C. Ko, C. Kao, L-J. Ho, K.
B. J. M. Cleutjens, J. Trapman, F. L. Graham, and L. W. K. Chung,
unpublished results).
 |
MATERIALS AND METHODS
|
---|
Cell Culture
LNCaP prostate cells were cultured as described (36). For
examination of androgen-driven promoter activity, the synthetic
androgen, R1881 (DuPont NEN, Boston, MA), was added to steroid-depleted
medium to a final concentration of 1 nM.
Construction of Plasmids
All plasmid constructs were prepared according to standard
procedures (37). The human androgen receptor expression plasmid pSVARo
and the LacZ-containing reporter plasmid pLACH were described
previously (38, 39). A mouse protamine gene fragment (mP1, +95 to +625,
see Ref. 24) provides the LacZ cassette with an intron and the
3'-untranslated region, including the polyadenylation signal.
PSA-61-LACH was generated by integration of the blunt ended
HindIII/HindIII (-6 kb/+12) fragment of the PSA
promoter into the SmaI site of the pLACH multiple cloning
site. PSA-4-LACH was generated by integration of the
EcoRI/HindIII (-632/+12 bp) PSA promoter
fragment into pLACH.
Transient Transfections
Cells were transfected according to the calcium phosphate
precipitation method, essentially as described (14).
Generation and Identification of Transgenic Mice
The 632-bp and 6-kb PSA promoter-driven LacZ genes were released
from vector sequences by restriction digestion, purified by gel
electrophoresis, and prepared for injection according to standard
methods (40). The appropriate fragments were microinjected into the
male pronuclei of fertilized eggs of C57BL6xDBA2C (F1) mice. The
presence of the transgene was established by PCR amplification on DNA
from tail biopsies (40), using oligonucleotide primers PSA-s:
5'-TTGTCCCCTAGATGAAGTCTCCATGA-3' and LacZ-as:
5'-CGCCAGGGTTTTCCCAGTCACGAC-3' (indicated in Fig. 1
).
Transgene copy numbers were quantitated by phosphoimage analyses of
Southern blots of tail DNA. To this purpose, 10 µg DNA were digested
with KpnI and SacI, electrophoresed on 0.8%
agarose gel, and transferred to Hybond N+ membrane
(Amersham, Cardiff, UK). Filters were hybridized at high stringency
with a random primed 32P-labeled protamine probe (see
RNA in Situ Hybridization). DNA transfer and filter
hybridization were carried out according to the protocol of the
manufacturer.
Liquid ß-Galactosidase Assay
ß-Galactosidase activity was measured in lysates of LNCaP
cells and mouse tissues using the Galacto-Light Plus chemiluminescent
reporter assay (Tropix Inc., Bedford, MA). Two to 5 mg of mouse tissue
were incubated in 100 µl lysis solution, and transfected LNCaP cells
were collected in 350 µl lysis solution. ß-Galactosidase activity
in 10 µl extract was corrected for variations in protein
concentrations (protein microassay, Bio-Rad, München,
Germany).
Whole Mount ß-Galactosidase Staining
Immediately after death, mouse tissues were fixed by perfusion
fixation in 2% paraformaldehyde in a 0.1 M piperazinebis
(ethane sulfonic acid) buffer (pH 6.9), containing 2 mM
MgCl2 and 1.25 mM EGTA. Tissues were dissected
and fixed for an additional 6090 min at room temperature. To
inactivate endogenous ß-galactosidase activity, tissues were washed
three times for 30 min in PBS (PBS, pH 8.6: 1.5 mM
KH2PO4/6.5 mM
Na2HPO4/2.7 mM KCl/135
mM NaCl). Subsequently, tissues were incubated in PBS for
60 min at 50 C. After cooling to room temperature, tissues were
incubated in prestaining solution (containing 2 mM
MgCl2, 5 mM K3Fe(CN)6,
5 mM K4Fe(CN)6, and 5
mM EGTA in PBS) for 60 min. After transfer to staining
solution (prestaining solution supplemented with 0.5 mg/ml X-Gal),
incubation was continued for 624 h at room temperature. The reaction
was stopped by extensive washing in PBS, and tissues were postfixed in
4% paraformaldehyde in PBS before paraffin embedding. Five-micrometer
sections were counterstained with neutral red.
RT-PCR
Isolation of total cellular RNA was carried out according to the
guanidinium isothiocyanate method (41). RT-PCR amplification of
LACZ-protamine (primers LACZ-s and PRO1/2-as), mouse kallikreins
(primers KALK- 3-s and KALK-4-as), mGK22 (mGK221/2-s and
mGK224/5-as), and GAPDH (GAPDH-s and GAPDH-as) were performed on 1
µg total RNA in the single tube Access RT-PCR* system
(Promega, Madison, WI), according to the protocol of the manufacturer.
Annealing steps were at 58 C, except for the kallikrein cDNAs expressed
in mouse prostate (primers KALK-3-s and KALK-4-as), which was at 50
C.
RT-PCR Primers
LACZ-s: 5'-AGCCATCGCCATCTG-3'
PRO1/2-as: 5'-GACGGCAGCATCTTCGCCTC-3' KALK-3-s:
5'-TGCGGATCCTCAGGCTGGGGCAGCA-3' KALK-4-as:
5'-TGTCAGATCTCCTGCACACAA/GCAT-3' mGK221/2-s:
5'-CTAGGAGGGATTGATGCTGC-3' mKG224/5-as:
5'-CCTCCTGAGTCTCCCTTACA-3' GAPDH-s:
5'-GGTCTACATGTTCCAGTATGACTCC-3' GAPDH-as:
5'-GAGACAACCTGGTCCTCAGTGTAGC-3'
The resulting PCR products were separated over a 2%
agarose gel and, in indicated cases, transferred to Hybond
N+ membrane. Filters were hybridized at high stringency
with random primed 32P-labeled probes specific for the
expected cDNA fragment. The PCR product obtained with primers KALK-3-s
and KALK-4-as was cloned in PCR-II (Invitrogen, Leek, The Netherlands),
and resulting clones were sequenced.
RNA in Situ Hybridization
Sense and antisense DIG-labeled protamine RNA probes were
generated on a 175-bp protamine cDNA fragment, obtained by RT-PCR on
mouse testis RNA with primers PRO-s (5' GAAGATGTCGCAGACGGAGG 3') and
PRO-as (5' GATGTGGCGAGATGCTCTTG 3'). The PCR fragment was first cloned
in pCR-II. After sequencing, the EcoRI-EcoRI cDNA
fragment was recloned in pTZ19 (Pharmacia, Uppsala, Sweden). After
linearization with HindIII, DIG-labeled RNA was transcribed
from the T7 promoter. Hybridization of 5-µm paraffin-embedded
sections and visualization with alkaline phosphatase-coupled anti-DIG
antibodies and indoxil-nitroblue tetrazolium substrate were done
essentially as described (42). Sections were counterstained with
neutral red.
Experimental Animals
In accordance with the NIH Guidelines for Care and Use of
Laboratory Animals, all experiments were conducted using the highest
standard for humane care.
 |
ACKNOWLEDGMENTS
|
---|
We thank J. Vaughan and Dr. G. Lozano (Department of Molecular
Genetics, MD Anderson Cancer Center, Houston, TX) for their
indispensable contribution to the generation of the transgenic animals.
We are indebted to Dr. F. T. Bosman for critical reading of the
manuscript, M. Vermey and C. M. A. de Ridder for skillful technical
support, H. Kock for animal care, and F. L. van der Panne for
photography.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. C. B. J. M. Cleutjens, Department of Pathology, Erasmus University, PO Box 1738, 3000 DR Rotterdam, The Netherlands.
This study was supported by a grant of the Dutch Cancer Society.
Received for publication March 20, 1997.
Revision received May 2, 1997.
Accepted for publication May 7, 1997.
 |
REFERENCES
|
---|
-
Catalona WJ, Smith DS, Ratcliff TL, Dodds KM, Coplen DE,
Yuan JJ, Petros JA, Andriole GL 1991 Measurement of prostate-specific
antigen in serum as a screening test for prostate cancer. N Engl
J Med 324:11561161[Abstract]
-
Oesterling JE 1991 Prostate specific antigen: a critical
assessment of the most useful tumor marker for adenocarcinoma of the
prostate. J Urol 145:907923[Medline]
-
Riegman PHJ, Vlietstra RJ, Klaasen P, van der Korput JAGM,
van Kessel AG, Romijn JC, Trapman J 1989 Characterization of the
prostate-specific antigen gene: a novel human kallikrein like gene.
Biochem Biophys Res Commun 159:95102[Medline]
-
Lundwall A 1989 Characterization of the gene for prostate
specific antigen, a human glandular kallikrein. Biochem Biophys Res
Commun 161:11511159[Medline]
-
Schedlich LJ, Bennets BH, Morris BJ 1987 Primary structure of
a human glandular kallikrein gene. DNA 6:429437[Medline]
-
Evans BA, Zhang XY, Close JA, Tregear GW, Kitamura N,
Nakanishi S, Callen DF, Baker E, Hyland VJ, Sutherland GR, Richards RI 1988 Structure and chromosomal localization of the human renal
kallikrein gene. Biochemistry 27:31243129[Medline]
-
Riegman PHJ, Vlietstra RJ, Klaasen P, van der Korput JAGM,
Romijn JC, Trapman J 1989 The prostate-specific antigen gene and the
human glandular kallikrein 1 gene are tandemly located on chromosome
19. FEBS Lett 247:123126[CrossRef][Medline]
-
Riegman PHJ, Vlietstra RJ, Suurmeijer L, Cleutjens CBJM,
Trapman J 1992 Characterization of the human kallikrein locus. Genomics 14:611[Medline]
-
Henntu P, Liao S, Vikho P 1992 Androgens up-regulate the
human prostate-specific antigen messenger ribonucleic acid (mRNA), but
down-regulate the prostatic acid phosphatase mRNA in the LNCaP cell
line. Endocrinology 130:766772[Abstract]
-
Young CYF, Andrews PE, Montgomery BT, Tindall DJ 1992 Tissue-specific and hormonal regulation of human prostate-specific
glandular kallikrein. Biochemistry 31:818824[Medline]
-
Riegman PHJ, Vlietstra RJ, van der Korput JAGM, Brinkmann AO,
Trapman J 1991 The promoter of the Prostate-specific antigen gene
contains a functional androgen responsive element. Mol Endocrinol 5:19211930[Abstract]
-
Cleutjens KBJM, van Eekelen CCEM, van der Korput HAGM,
Brinkmann AO, Trapman J 1996 Two androgen response regions cooperate in
steroid hormone regulated activity of the prostate specific antigen
promoter. J Biol Chem 271:63796388[Abstract/Free Full Text]
-
Schuur ER, Henderson GA, Kmetec LA, Miller JD, Lamparski
HG, Henderson DR 1996 Prostate-specific antigen expression is regulated
by an upstream enhancer. J Biol Chem 271:70437051[Abstract/Free Full Text]
-
Cleutjens KBJM, van der Korput HAGM, van Eekelen CCEM, van
Rooij HCJ, Faber PW, Trapman J 1997 An androgen response element in a
far upstream enhancer region is essential for high, androgen-regulated
activity of the prostate-specific antigen promoter. Mol Endocrinol 11:148161[Abstract/Free Full Text]
-
Evans BA, Drinkwater CC, Richards RI 1987 Mouse glandular
kallikrein genes. Structure and partial sequence analysis of the
kallikrein gene locus. J Biol Chem 262:80278034[Abstract/Free Full Text]
-
Watt KWK, Lee P-J, MTimkulu T, Chan W-P, Loor R 1986 Human
prostate-specific antigen: structural and functional similarity with
serine proteases. Proc Natl Acad Sci USA 83:31663170[Abstract]
-
Brady JM, Wines DR, MacDonald RJ 1989 Expression of two
kallikrein gene family members in the rat prostate. Biochemistry 28:52035210[Medline]
-
Wang MC, Papsidero LD, Kuriyama M, Valenzuela LA, Murphy GP,
Chu TM 1981 Prostate antigen: a novel marker for prostatic cancer.
Prostate 2:8996[Medline]
-
Goldfarb DA, Stein BS, Shamsadeh M, Petersen RO 1986 Age-related changes in tissue levels of prostatic acid phosphatase and
prostate specific antigen. J Urol 136:12661269[Medline]
-
Fahnestock M, Woo JE, Lpoez GA, Snoe J, Walz DA, Arici MJ,
Mobley WC 1991 Beta-NGF-endopeptidase: structure and activity of a
kallikrein encoded gene mGK-22. Biochemistry 30:34433450[Medline]
-
Kim WS, Nakayama K, Nakagawa T, Haragushi K, Murakami K 1991 Mouse submandibular gland prorenin-converting enzyme is a member
of glandular kallikrein family. J Biol Chem 266:1928319287[Abstract/Free Full Text]
-
Drinkwater CC, Evans BA, Richards RI 1987 Mouse glandular
kallikrein genes: Identification and characterization of the genes
encoding the epidermal growth factor binding proteins. Biochemistry 26:67506756[Medline]
-
Zarghami N, Diamandis EP 1996 Detection of prostate-specific
antigen mRNA and protein in breast tumors. Clin Chem 42:361366[Abstract/Free Full Text]
-
Peschon JJ, Behringer RR, Brinster RL, Palmiter RD 1987 Spermatid-specific expression of protamine 1 in transgenic mice. Proc
Natl Acad Sci USA 84:53165319[Abstract]
-
Young DC, Kingsley SD, Ryan KA, Dutko FJ 1993 Selective
inactivation of eukaryotic beta-galactosidase in assays for inhibitors
of HIV-1 TAT using bacterial beta-galactosidase as a reporter enzyme.
Anal Biochem 215:2430[CrossRef][Medline]
-
Schaffner, DL, Barrios R, Shaker MR, Rajagopalan R, Huang SL,
Tindall DJ, Young CYF, Overbeek PA, Lebovitz RM, Lieberman MW 1995 Transgenic mice carrying a PSArasT24 hybrid gen develop
salivary gland and gastrointestinal tract neoplasms. Lab Invest 73:283290
-
vanKrieken JH 1993 Prostate marker immunoreactivity in
salivary gland neoplasms. Am J Surg Pathol 17:410414[Medline]
-
Edmondson DG, Roth SY 1996 Chromatin and transcription. FASEB
J 10:11731182[Abstract/Free Full Text]
-
Southard-Smith M, Lechago J, Wines DR, MacDonald RJ, Hammer RE 1992 Tissue-specific expression of kallikrein family transgenes in mice
and rats. DNA Cell Biol 11:345358[Medline]
-
Buttyan R, Slawin K 1993 Rodent models for targeted
oncogenesis of the prostate gland. Cancer Metast Rev 12:1119[Medline]
-
Allison J, Zhang Y-L, Parker MG 1989 Tissue specific and
hormonal regulation of the gene for rat prostatic steroid binding
protein in transgenic mice. Mol Cell Biol 9:22542257[Medline]
-
Maroulakou IG, Anver M, Garret L, Green JE 1994 Prostate and
mammary carcinoma in transgenic mice carrying a rat C3(1) simian virus
40 large tumor antigen fusion gene. Proc Natl Acad Sci USA 91:1123611240[Abstract/Free Full Text]
-
Greenberg NM, DeMayo FJ, Sheppard PC, Barrios R, Lebovitz R,
Finegold M, Angelopoulou R, Dodd JG, Duckworth ML, Rosen JM, Matusik R 1994 The rat probasin promoter directs hormonally and developmentally
regulated expression of a heterologous gene specifically to the
prostate of transgenic mice. Mol Endocrinol 8:230239[Abstract]
-
Greenberg NM, DeMayo F, Finegold MJ, Medina D, Tilley WD,
Aspinall JO, Cunha GR 1995 Prostate cancer in a transgenic mouse.
Proc Natl Acad Sci USA 92:34393443[Abstract]
-
Gingrich JR, Barrios RJ, Morton RA, Boyce BF, DeMayo FJ,
Finegold MJ, Angelopoulou R, Rosen JM, Greenberg NM 1996 Metastatic
prostate cancer in a transgenic mouse. Cancer Res 56:40964102[Abstract]
-
Horoszewicz JS, Leong SS, Kawinski E, Karr JP, Rosenthal H,
Ming Chu T, Mirand EA, Murphy GP 1983 LNCaP model of human prostatic
carcinoma. Cancer Res 43:18091818[Abstract]
-
Sambrook, J, Fritsch, EF, Maniatis, T 1989 Molecular Cloning:
A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY
-
Brinkmann AO, Faber PW, van Rooij HCJ, Kuiper GGJM, Ris C,
Klaassen P, van der Korput JAGM, Voorhorst MM, van Laar JH, Mulder E,
Trapman J 1989 The human androgen receptor: domain structure, genomic
organization and regulation of expression. J Steroid Biochem 34:307310[CrossRef][Medline]
-
Bou-Gharios G, Garret LA, Rossert J, Niederreither K,
Eberspaecher H, Smith C, Black C, deCrombrugghe B 1996 A potent
far-upstream enhancer in the mouse pro alpha 2 (I) collagen gene
regulates expression of reporter genes in transgenic mice. J Cell Biol 134:13331344[Abstract]
-
Hogan B, Beddington R, Costantini F, Lacy E 1994 Manipulating
the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY
-
Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ 1979 Isolation of biological active ribonucleic acid from sources
enriched in ribonuclease. Biochemistry 18:52945299[Medline]
-
DeBlock M, Debrouwer D 1993 RNA-RNA in situ
hybridization using digoxigenin-labeled probes: the use of
high-molecular-weight polyvinyl alcohol in the alkaline phosphatase
indoxyl-nitroblue tetrazolium reaction. Anal Biochem 215:8689[CrossRef][Medline]