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The Prostate 32:16–26 (1997)
Abnormal Prostate Development in C3(1)-bcl-2
Transgenic Mice
Xuejun Zhang,1 Min-Wei Chen,1 Albert Ng,1 Po-Ying Ng,1 Chung Lee,3
Mark Rubin,2 Carl A. Olsson,1 and Ralph Buttyan1,2*
1
Department of Urology, College of Physicians and Surgeons, Columbia University,
New York, New York
2
Department of Pathology, College of Physicians and Surgeons, Columbia University,
New York, New, York
3
Department of Urology, Northwestern University School of Medicine, Chicago, Illinois
BACKGROUND. Recent hypotheses to explain the etiology of abnormal growth associated
with prostate disease have invoked perturbations in the rate of apoptosis as an important
contributor to the onset and progression of these diseases. For this reason, the apoptosis
suppressing oncoprotein bcl-2 has come under scrutiny with regards to its role in prostate
diseases. In order to evaluate the role of bcl-2 in human prostate disease and to develop an
animal model to test anti-bcl-2 therapies, we generated transgenic mice in which bcl-2 expression is targeted to the mouse prostate gland.
METHODS. Mouse embryos were microinjected with recombinant DNA constructed by
fusing a modified rat C3(1) promotor element to cDNA encoding human bcl-2. Presence of the
C3(1)-bcl-2 transgene in progeny was identified by Southern blot and polymerase chain
reaction (PCR) analysis. RNase protection assays were used to analyze RNA from 15 organs
of these mice. Western blot assays and immunohistochemical staining were used to confirm
the tissue-specific protein expression of human bcl-2 and its cellular localization.
RESULTS. Three lines of C3(1)-bcl-2 transgenic mice were established. Founder mice carried
2–20 copies of the transgene. Expression of human bcl-2 from the transgene was limited to the
prostate gland and testis of males as well as the uterus of females. In the prostate gland,
human bcl-2 protein was found only in prostatic epithelial cells. Microscopic analysis of
prostate glands from individual males (three lines) showed that these glands were often
abnormal, with increased accumulation of cells in the prostatic stroma as well as the epithelium.
CONCLUSIONS. These transgenic mice appear to provide a novel animal model for studying neoplastic development of the prostate, with particular emphasis on the bcl-2 protein and
the role of apoptosis regulation in such development. Prostate 32:16–26, 1997.
© 1997 Wiley-Liss, Inc.
KEY WORDS:
prostate gland; apoptosis; bcl-2; transgenic mice; prostate-steroid-binding
protein
INTRODUCTION
Benign and malignant growth diseases of the prostate gland rank among the most prevalent afflictions
associated with human aging [1,2]. The causative
agents involved in the abnormal onset of prostate
gland growth in adults are poorly understood. One of
the major roadblocks to progress in understanding
© 1997 Wiley-Liss, Inc.
prostate growth disease has been the lack of animal
models for research. Other than humans, dogs are the
*Correspondence to: Ralph Buttyan, Department of Urology, College of Physicians and Surgeons, Columbia University, 630 W. 168th
Str., New York, NY 10032.
Received 7 February 1996; Accepted 18 June 1996
Abnormal Prostates in Transgenic Mice
only other animal known to naturally develop prostate hyperplasia with a high frequency [3]. Attempts
to establish rodent models of prostatic hyperplasia
and malignancy using chemical carcinogenic agents
have been reported [4,5], but these animal models are
often not suitably reproducible or sufficiently reliable
to initiate appropriate studies. More recently, transgenic procedures involving the direct genetic manipulation of mouse embryos with recombinant DNA vectors have produced some lines of mice in which the
prostate-directed expression of viral genes has been
shown to induce hyperplasia and malignancy [6,7].
We report on the utilization of transgenic technology
to produce several independent lines of mice in which
the human bcl-2 gene is selectively expressed in the
mouse prostate gland.
The rationale for the selection of bcl-2 for such experiments comes from a number of recent reports
identifying a potential relationship between bcl-2 expression and neoplastic development of the human
prostate gland. Immunohistochemical analyses of human prostate tissues showed that bcl-2 protein was
not detectable in normal human prostatic secretory
epithelial cells, but was highly expressed in prostatic
intraepithelial neoplasias (PIN), as well as in a minor
subset of untreated prostatic adenocarcinomas and in
the vast majority of prostate adenocarcinomas obtained from patients subsequent to hormonedeprivation therapy [8,9]. Furthermore, transfection of
the prostate cancer cell line, LNCaP, with a bcl-2 expression vector makes this normally androgensensitive cell line resistant to the effects of androgendeprivation in vitro and when implanted into nude
mice [10]. Given this descriptive relationship between
increased bcl-2 expression and the development and
progression of prostatic cancer to the hormoneresistant state, we wanted to determine the effects of
bcl-2 overexpression on prostate gland development
in a laboratory animal model.
Previous studies showed that the 58 DNA promotor
region of the rat ventral prostate secretory protein
gene product, C3(1), is suitable for targeting the expression of a chimeric reporter gene (b-galactosidase
or SV-40 T-antigen) to the prostate glands of transgenic mice [6,11]. In these earlier experiments, however, the unmodified C3(1) promotor region also permitted promiscuous expression of the reporter gene in
other mouse tissues. For this study, we further modified the rat C3(1) promotor region that was utilized for
generation of transgenics in the hopes of obtaining a
more specific targeting of the mouse prostate gland in
transgenic progeny. The three genetic modifications
that were made to the C3(1) promotor element included: 1) increasing the length of the 58 promotor
region utilized; 2) retention of the first exon and intron
17
of C3(1); and 3) the introduction of site-specific mutations in the two potential translation initiation codons
within the first exon. This genetically-modified promotor was fused to the coding region of human bcl-2
cDNA, and this construct was utilized to create several independent lines of transgenic mice.
MATERIALS AND METHODS
Construction of Chimeric C3(1)-bcl-2 Transgene
A 6.4-kb BamHI-PstI restriction fragment containing
4.1 kbp of the upstream 58 promotor region through 17
bp of the second exon of C3(1) were obtained from the
C3(1) genomic plasmid p611 (a gift from Dr. M.
Parker, Imperial Cancer Research Fund, London, UK)
[12,13]. This fragment was subcloned into the pALTER-1 plasmid vector (Promega, Madison, WI) for in
situ mutagenesis. Since the first exon for C3(1) contains the authentic ATG translation start codon as well
as a second out-of-frame ATG, these two potential
ATG codons were mutated to ACGs by the Altered
Sites In Vitro Mutagenesis Systemt (Promega). Briefly,
this system requires the use of at least two mutagenic
oligonucleotides, one to induce a reversion in the mutated ampicillin resistance gene of pALTER, and another to induce a selected mutation within the DNA
fragment inserted into pALTER. For our modifications, we needed to place two independent sitespecific mutations in the C3(1) promotor fragment,
and this was done by utilizing three mutagenic oligonucleotides, one provided with the kit (to revert the
ampr gene of pALTER), and two additional ones (synthesized by National Biosciences, Inc., Plymouth, MN)
to insert mutations at the sequential ATG sites in the
first exon of C3(1), i.e., 58-GCCTCAACACGAAGCTGG-38 and 58-TGCTGCTACGCCAGTGGTAA-38.
These oligonucleotides were simultaneously annealed
to the single-stranded DNA template of the pALTER
vector. The annealed DNA was treated with T4 DNA
polymerase to extend the primers and was then ligated with T4 DNA ligase. The treated vector was
used to transform competent BMH 71-18 cells, and
bacteria-containing mutated vectors were selected by
growth on ampicillin agar plates. Resistant colonies
were picked and the plasmids were sequenced by use
of standard dideoxynucleotide methods. A colony that
contained both mutations within the first exon was
selected for further work. The modified C3(1) promoter from this colony was subcloned into BamHI-PstI
digested pBluescript SK vector (Stratagene, La Jolla,
CA). The DNA insert from the pSFFV/bcl-2 plasmid
(obtained from Dr. S. Korsmeyer, Washington University School of Medicine, St. Louis, MO) [14] containing
the 1.8-kb human bcl-2 cDNA, 0.9-kb SV-40 early
18
Zhang et al.
splice, and SV-40 polyadenylylation signal was excised with EcoRI (partial digestion)-HindIII and subcloned into the downstream EcoRI-HindIII (partial
digestion)-digested C3(1)-pBluescript vector. This
construct was called C3(1)-bcl-2, and the correct transcriptional orientation of the bcl-2 reporter gene was
confirmed by DNA sequencing by use of standard
dideoxynucleotide methods.
Experimental Animals and Production of
Transgenic Mice
Animals used in these experiments were maintained within the Institute of Comparative Medicine at
the Columbia University Health Sciences Division. All
experiments were conducted in accordance with the
highest standards of humane animal care, as outlined
in the NIH Guide for the Care and Use of Laboratory
Animals. Transgenic mice were generated as described [15]. The entire C3(1)-bcl-2 transgene was excised from the vector with NotI and ClaI. The fragment
was isolated by gel electrophoresis followed by electroelution from the gel, and was further purified by
CsCl ultracentrifugation. Purified DNA was resuspended in microinjection buffer (10 mM Tris-HCl, pH
8.0, 0.1 mM EDTA) to a final concentration of 2–5 ng/
ml and used for microinjection into the male pronucleus of fertilized mouse oocytes derived from the
C57BL/6J × CBA/J mouse strain (Jackson Laboratory,
Bar Harbor, ME). Microinjected embryos were maintained in culture for 24 hr prior to implantation into
pseudopregnant female recipients. Litters were born
to these mice 21 days later, and the progeny were
maintained with the surrogate mothers until weaning.
Portions of the tail were clipped at this time for extraction of DNA.
Identification of Transgenic Mice
Transgenic progeny were identified by Southern
blot analysis of tail DNA isolated from 3-week-old
litters using standard techniques [16]. Ten micrograms
of tail DNA were digested with EcoRI and electrophoresed through a 1% agarose gel. The DNA was transferred to nylon membranes (Boehringer Mannheim,
Inc., Indianapolis, IN) and hybridized with the 1.8-kb
human bcl-2 cDNA probe labeled with 32P-dCTP, using the Random Primed DNA Labeling Kitt (Boehringer Mannheim, Inc.). The membranes were washed
under high stringency conditions, and were exposed
to X-ray film at −70°C overnight. Transgenic progeny
were identified by the presence of a 1.8-kb human
bcl-2 cDNA band, and the number of copies was calculated by comparing the film band density obtained
from the mouse endogenous bcl-2 gene with the
intensity of the human bcl-2 band performed with the
Molecular Dynamics Scanning Laser Densitometer
(Molecular Dynamics, Sunnyvale, CA). A PCR-based
technique was then used to confirm positive offspring
transgenic mice, as previously described [17]. The
primers used in PCR screening were designed to fall
within the C3(1) first intron (A) and human bcl-2
cDNA (B) (detailed in Fig. 1). The sequences of the
synthetic oligonucleotides were as follows: A, 58GCCCATCACCTTGCTTAT-38; B, 58-CACATCTCCCGCATCCCACT-38. These primers produce a 283-bp
DNA fragment following PCR amplification of the recombinant C3(1)-bcl-2 transgene.
RNA Isolation and Analysis
Total cellular RNA was isolated from transgenic
mouse organs including brain, heart, kidney, spleen,
lung, thymus, salivary, bladder, gut, prostate, seminal
vesicles, testis, muscle, uterus, and mammary gland,
using the RNzole B reagent (TelTest, Inc., Friendswood, TX), following the manufacturer’s instructions.
RNase protection assays were performed on these
RNAs as previously described [18], with the RPA II
Ribonuclease Protection Assay Kitt (Ambion, Inc.,
Austin, TX). A small cDNA fragment of 283 nucleotides, encoding a portion of the C3-bcl-2 transgene,
was obtained by PCR amplification from the intact
C3(1)-bcl-2 plasmid, as mentioned above. The amplified DNA fragment was cloned into the TA II cloning
vector (Invitrogen, San Diego, CA), and was sequenced using standard dideoxynucleotide methods.
This fragment will protect a chimeric mRNA sequence
containing the 17 nucleotides from C3(1) exon 2 and
the adjacent 163 nucleotides from human bcl-2 cDNA
(see Fig. 1). Synthesis and purification of the 32Plabeled antisense riboprobe from this vector were
done as previously described [18]. Twenty micrograms of total RNA were denatured and hybridized
overnight at 45°C to labeled antisense RNA transcribed in vitro in the presence of 32P-UTP. RNase
treatment was performed at 37°C for 1 hr. Digests
were applied to 8% acrylamide sequencing gels and
electrophoresed at 350 V for 3 hr. The gel was exposed
overnight to X-ray film to produce an autoradiograph.
Western Blot Analysis
Transgenic and normal control mouse prostate, testis, and uterus removed from necropsy were frozen in
liquid nitrogen, powdered, and homogenized in lysis
buffer (50 mM Tris-HCl, pH 8.0, 150 mM sodium chloride, 0.1% sodium dodecyl sulfate, 1% NP-40, and
0.5% sodium deoxycholate) on ice. Proteinase inhibitors, including PMSF (100 mg/ml), pepstatin A (1 mg/
ml), aprotinin (1 mg/ml), and leupeptin (1 mg/ml)
Fig. 1. Top: Diagrammatic description of DNA elements involved in construction of the recombinant C3(1)-bcl-2 gene. A genetically modified (mutated) fragment containing 4.1 kbp
DNA upstream from the transcriptional start site of C3(1), as well as the first exon, first intron, and a small portion (17 bp) of the second exon, were fused to a 1.85-kbp cDNA fragment
of human bcl-2 [14] and the early splice site and poly A signal site for SV-40. Bottom: Partial sequence of exon 1 of C3(1), showing two sites where potential translation start codons
(ATGs) were mutated to ACGs by an in situ mutagenesis system. a and b (top) represent approximate sites for binding of PCR primers, as described in Materials and Methods.
20
Zhang et al.
(Sigma Chemical Co., St. Louis, MO) were added to
the lysis buffer prior to homogenization. Insoluble debris was removed by centrifugation at 10,000g for 10
min at 4°C. Protein concentrations were determined in
these extracts using the Bio-Rad Protein Assay System
(Bio-Rad Labs, Inc., Richmond, CA). Aliquots of cell
extracts containing 50 mg of protein were electrophoresed on an 8% Laemmli SDS-polyacrylamide gel at
150 V for 45 min and electrophoretically transferred to
a nitrocellulose filter (Amersham Life Science, Arlington Heights, IL) at 110 V for 60 min in 25 mM TrisHCl, pH 8.0, 0.192 M glycine, and 20% methanol. The
filter was blocked in TBS-T buffer (20 mM Tris-HCl,
pH 8.0, 0.136 M NaCl, 5% nonfat milk, and 0.5%
Tween-20) at 4°C for 1 hr and then incubated overnight with a mouse monoclonal anti-human-bcl-2 antibody (Dako bcl-2, 124; Dako Corp., Carpinteria, CA)
[10] diluted to 1:2,000 in TBS-T. After a series of
washes in TBS-T buffer, the filter was incubated with
the secondary antibody (sheep anti-mousehorseradish peroxidase complex), supplied by Amersham Life Science, Inc. in the ECL Western Blotting
Analysis System (Amersham Life Science, Inc.). Chemiluminescent detection of antibody was accomplished using the reagents provided in this system,
following exposure of the blot to Kodak XAR-5 X-ray
film (Eastman Kodak, Rochester, NY) for 15 sec.
Immunohistochemical Studies of Transgenic Mice
Tissues of prostate, testis, and uterus from nontransgenic control and transgenic mice were collected
at necropsy and fixed in 10% neutral buffered formalin. The tissue was dehydrated and embedded in paraffin, and 5-m thin sections were cut from these tissues
with a microtome. The sections were deparaffinized
with xylene and rehydrated in a graded series of ethanol solutions. The immunostaining analysis for transgenic human bcl-2 protein was performed with the
HistoMouse Kit (Zymed Laboratories, Inc., South San
Francisco, CA), which was designed to detect reactivity of mouse primary antibodies on rodent tissue without background. Briefly, tissue sections were treated
with 3% peroxidase quenching solution for 10 min.
Nonspecific background was eliminated by incubating the slides with blocking solution A and blocking
solution B. The mouse monoclonal anti-human bcl-2
antibody (Dako 124, Dako Corp.) was incubated on
the tissues, followed by addition of a biotinylated secondary antibody (reagent 1C). Streptavidinperoxidase was then added (reagent 2) to bind to the
biotin residue on the linking antibody. The presence of
peroxidase was revealed by addition of substratechromogen solution (reagents 3A–C). Then peroxide
was utilized to convert the substrate to a red insoluble
deposit, which demonstrates the location of the transgenic human bcl-2 protein.
Histology
Tissues for light microscopy were collected from
sacrificed animals. Some tissues were fixed and embedded, as described above for preparation of thin
sections, while other tissues were embedded in OCT
compound and rapidly frozen to −70°C in a biopsy
mold. Thin sections (8 m) were obtained from the frozen tissues by a cryostat. All sections were stained
with hematoxylin and eosin for microscopic analysis.
RESULTS
Generation of C3-bcl-2 Transgenic Mice
C3(1) is a rat gene encoding the C3 peptide subunit
of the major secretory protein of the ventral prostate
gland, PSP [19]. Previously, we reported some success
in targeting b-galactosidase (b-gal) expression to the
prostate glands of transgenic mice with the use of a
chimeric DNA molecule that was constructed by fusing a portion of the 58 promotor region of the rat C3(1)
gene to the coding region for bacterial b-gal [11]. In
some founder lines made with this DNA construct, the
C3(1) promotor also allowed promiscuous expression
of the b-gal reporter in the seminal vesicles as well as
in the testis. Likewise, another report describing the
use of a C3(1) promotor region to target SV-40 Tantigen expression to the prostate gland also identified promiscuous expression of the transgene product
in thyroid, salivary gland, and cartilage, as well as in
the breast tissue of females [6]. In an attempt to further
restrict the tissue-specific expression of a reporter
gene, we first made genetic modifications to the C3(1)
promotor element (detailed in Fig. 1) that was subsequently utilized to target the expression of a human
bcl-2 reporter gene to the mouse prostate gland. The
modifications that we made increased the amount of
58 upstream (C3(1) promotor) DNA and ensured the
retention of any potential genetic regulatory element
within the first intron of rat C3(1), an area containing
a putative site for the presence of an androgenreceptor binding and response element [20]. The promoter sequences of C3(1) that were utilized in our
experiments are contained within a DNA fragment
generated by BamHI and PstI digestion of the C3(1)
genomic clone, 611 [12,13]. This fragment contains 4.1
kbp of the 58-flanking C3(1) promoter sequence, as
well as the first exon, entire first intron, and a small 58
fragment of exon 2. The two potential ATG translation
initiation sites within the first exon were sequentially
mutated (to ACG) to prevent inappropriate translation
Abnormal Prostates in Transgenic Mice
21
specimens examined. For mice in which the transgene
was integrated into genomic DNA, we also expected
to find hybridization to a fragment of 1.85 kbp. This
fragment was detected in digested tail DNA extracted
from progeny animals 3, 5, 15, 19, and 21 (Fig. 3A,
lanes 3–7). No hybridization to a 1.85-kb fragment was
detected in the DNA extracted from a control mouse
(Fig. 3). Additional and distinct hybridizing bands
were found in DNAs obtained from mice 3, 5, 19, and
21, demonstrating that the transgenic bcl-2 was inserted at separate sites of the mouse genome in these
lines. A comparison of the transgenic bcl-2 band intensities to the intensity of the normal mouse genomic
bcl-2 gene suggests that about 2–20 copies were incorporated in the various transgenic progeny lines. A
PCR amplification technique was also used to screen
DNA from the putative positive transgenic mice. All 5
transgenic founder mice demonstrated the expected
283-bp transgene PCR product (Fig. 3B).
Tissue-Specific Expression of Human bcl-2 in
Transgenic Mice
Fig. 2. Autoradiograph of DNA sequencing gel demonstrates
introduction of mutations in the two ATG sites within the first
exon of C3(1) prior to construction of the C3(1)-bcl-2 transgene.
In this manipulation, ATG sites were mutated to ACGs.
of the recombinant transgene message by means of in
situ mutagenesis. DNA sequencing was used to confirm that both the authentic ATG translation start
codon and the out-of-frame ATG sites were mutated
to ACGs in the final modified promotor fragment (Fig.
2). The transgene of C3-bcl-2 was completed by enzymatic ligation of DNA fragments, including the modified 58 C3(1) promoter, human bcl-2 cDNA, and the 38
SV-40 polyadenylation signal (Fig. 1). The hybrid
C3(1)-bcl-2 gene fragment was purified by CsCl ultracentrifugation and was microinjected into the pronuclei of C57BL/6 × C57BL/F1 fertilized eggs. The twocell-stage embryos were reimplanted into pseudopregnant C57BL/6 outbred females, and the females
were maintained through the birth and subsequent
weaning of the pups.
Analysis of DNA obtained from tail biopsies of the
pups identified 5 potential founder mice by Southern
blot analysis. Tail DNAs were digested with EcoRI restriction endonuclease, electrophoresed, and blotted
onto nitrocellulose paper. The blot was hybridized to
a radiolabeled 1.85-kbp human bcl-2 cDNA probe
(Fig. 3A). Two EcoRI bands (5.7 kb and ∼10 kb), corresponding to hybridization with the endogenous
mouse bcl-2 gene fragments, were seen in all DNA
To test the potential of the modified C3(1) promotor
fragment for targeting prostate gland-specific expression of bcl-2, we analyzed the RNA of numerous tissues obtained from first-generation (heterozygous)
transgenic offspring for expression of hybrid C3-bcl-2
sequences by an RNase protection assay. An antisense
riboprobe that would protect transgene mRNA containing 17 nucleotides of C3(1) exon 2 fused to 163
nucleotides of human bcl-2 cDNA was used to detect
the presence of this messenger RNA in various organs
(see Fig. 4A). This riboprobe is expected to protect
(following hybridization) a 180-nucleotide fragment
from RNase digestion when the transgene is expressed. The 180-nucleotide-protected bands were detected when test RNAs were obtained from the prostate glands and testis of male progeny mice as well as
from the uterus of female progeny mice in three different transgenic mouse lines (3, 5, and 21). An example of tissues (male and female) analyzed from
mice in the second generation of line 5 is shown in the
autoradiograph of Figure 4B. No protected fragments
were obtained when RNAs were obtained from tissues
(including prostate, uterus, and testis) of nontransgenic littermate mice (Fig. 4B). Expression of human
bcl-2 protein was confirmed in these same tissues of
three transgenic lines by Western blot analyses. In Figure 5, a Western blot probed with a monoclonal antibody against human bcl-2 protein revealed the presence of the 26-kd bcl-2 protein in an extract of the
derivative LNCaP cell line (LNCaP-bcl-3) that overexpresses bcl-2 subsequent to transformation [10], as
well as in protein extracts obtained from the prostate
22
Zhang et al.
Fig. 3. A: Autoradiograph of Southern blot demonstrates hybridization of bcl-2 cDNA to a 1.85-kbp DNA fragment present in transgenic
mouse founder lines, as well as to the endogenous mouse bcl-2 gene fragments at 10 kbp and 5.2 kbp. B: Ethidium bromide staining pattern
of an agarose gel following electrophoresis of reaction products obtained from a PCR amplification reaction using DNA obtained from a
control (nontransgenic) mouse tail (lane 1), or from DNAs extracted from the tails of founder mouse lines, as indicated. A DNA fragment
at 283 bp was generated from amplification of the chimeric C3(1)-bcl-2 gene.
glands, testis, and uterus of transgenic mice (lines 3, 5,
and 21). The bcl-2 protein was abundantly expressed
in these tissues but was absent from the comparable
tissues obtained from nontransgenic mice (Fig. 5).
Immunohistochemical Analysis of Transgenic
Mouse Tissues
A mouse monoclonal antibody against human bcl-2
was used to immunostain prostate, testis, and uterus
from heterozygote transgenic progeny (lines 3, 5, and
21). This antibody distinctly stained epithelial cells of
the ventral prostate (Fig. 6), as well as interstitial (fibroblastic) cells of the testis and uterus (not shown).
Equivalent application of this staining protocol to sections of these same tissues obtained from control (nontransgenic) littermates did not identify any immunostaining cells in these tissues.
Abnormalities of Prostate Gland Detected in
Transgenic Progeny Mice
Overall, the size and weights of prostate glands (as
well as the other male urogenital tract organs) of heterozygote transgenic male mice were virtually indistinguishable from tissues obtained from nontrans-
genic, age-matched males. Nor was there any evidence for increase in the proliferative index of these
tissues, based on overt counting of mitotic cells in thin
sections of tissues. Nonetheless, the prostate glands
obtained from some of these mice showed a peculiar
morphological appearance when examined by microscopy. As shown in Figure 7, a ventral prostate gland
isolated from one particular transgenic male mouse
(line 5, 3 months old) was distinctly affected, having
an extensive accumulation of cells both within the
stromal compartment as well as in the epithelial cell
compartment. In spite of the fact that the prostate
gland was not significantly enlarged compared to normal mouse prostate gland, microscopic analysis
showed that the cellular elements were crowded to the
extent that very little luminal space was evident in the
prostatic ducts. Ventral prostate tissues from 6 of 14
other individual males of this line were also affected to
a lesser degree, as were 3 of 9 males analyzed from
line 21, and 1 of 5 males analyzed from line 3. The
frequency with which this phenotype is observed in
males of the different lines (line 5 > line 21 > line 3)
reflects the relative ranking of these lines for transgene
copy numbers (∼20 copies, line 5; ∼4 copies, line 21;
and ∼2 copies, line 3). In summary, prostate tissues
obtained from individual transgenic males from any
Abnormal Prostates in Transgenic Mice
23
Fig. 4. A: Diagram identifies potential fragment of C3-bcl-2 transgene RNA that will be protected by antisense riboprobe generated by
in vitro transcription of a DNA fragment produced by PCR, utilizing primers a and b. B: RNase protection assay identifies expression of
the chimeric C3(1)-bcl-2 gene in tissues obtained from 4-month-old transgenic mice from first-generation heterozygotes of line 5. The
180-bp protected fragment was observed in RNA extracted from the prostate, testis, and uterus of this transgenic line, but not in RNA
extracted from the same tissues of nontransgenic (control) mice.
given line showed variation in this characteristic. Almost half the males from line 5 were affected, whereas
a decreasing proportion of males from lines 21 and 3
(having fewer copies of the transgene) showed signs
of this atypical prostatic hypertrophy. For mice that
were affected, prostatic hypertrophy involved both
stromal and epithelial elements, and this is enigmatic
considering that the transgene product (human bcl-2)
is only expressed in the epithelial cells. The two other
tissues that consistently demonstrated expression of
human bcl-2, testis and uterus, had no discernible phenotypic change detectable by size or microscopic
analysis in any of the male or female progeny.
DISCUSSION
The bcl-2 protein is a potent suppressor of apoptosis [21,22]. This protein is well-known for its role in the
development of certain forms of human lymphomas
[23]. Likewise, it is suspected of having a natural role
in the development and progression of cancers of the
human breast and prostate gland [8,9,24]. The transgenic mice which we produced in these experiments,
having greatly elevated amounts of human bcl-2 protein expressed in the prostate gland, should help in
identifying any potential neoplastic or malignant phenotype associated with bcl-2 overexpression in the
prostate.
In our experiments, targeting of bcl-2 protein expression to the mouse prostate gland in transgenic
animals was accomplished by use of a geneticallymodified promotor element from the rat C3(1) gene.
These modifications included an increase in the
amount of C3(1) DNA upstream from the 58 transcription start site utilized for the recombinant transgene.
Previously, we had used a smaller promotor element
24
Zhang et al.
Fig. 5. Immunochemical analysis of a Western blot identifies the
25-kd human bcl-2 protein in extracts of prostate tissue, testis,
and uterus obtained from first-generation heterozygotes of line 5,
but not from comparable tissue obtained from nontransgenic
mice.
derived from this gene to target the expression of a
neutral reporter gene (b-gal) and had found that the 58
C3(1) promotor allowed for promiscuous expression
of the reporter gene in other male tissues (testis and
seminal vesicle) [11]. Likewise, in another study in
which the C3(1) promotor was utilized to target the
expression of SV-40 T-antigen to the prostate, promiscuous expression of the T-antigen was found in a
number of other transgenic mouse tissues [6]. Secondly, because genetic regulatory elements (an androgen receptor binding site) were also described in the
first intron of the C3(1) gene [20], we had hoped that
genetic manipulations of this 58 C3(1) promotor, so
that the first intron would be maintained, might increase the specificity of targeting reporter genes to the
prostate gland in transgenic mice. Since the first intron
contains two ATG codons (potential translation start
sites), these sites were mutated to ACGs so that translation of the reporter gene must be initiated by an
appropriate codon within the reporter cDNA. In the
three independent lines of transgenic mice that we
have so far characterized, the modified C3(1) promotor element containing the first exon and intron still
allows for promiscuous expression of the bcl-2 reporter gene (in our lines, to the interstitial cells of the
testis and uterus). The reason for this promiscuous
expression is not clear, since C3(1) mRNA or protein is
not normally expressed in mouse testis or uterus [19].
The promiscuous expression of the bcl-2 reporter protein, however, does not seem to overtly affect the phenotype of the testis or uterus of the transgenic mouse
lines in the same way as the prostate gland. Perhaps
this is because testicular and uterine fibroblastic cells
have such a normally long life span (and low cell turnover) that the increased expression of bcl-2 here does
not further affect the tissue.
To date, a minor proportion (20–50%) of male heterozygous progeny mice from at least three different (independent) lines of C3(1)-bcl-2 transgenic mice has shown
benign morphological changes in the prostate glands,
involving increased cellular content. This phenotype is
variable, appearing more frequently in lines with a
higher transgene copy number, and with differing severity in individual mice. The affect was most pronounced in males from line 5, and it appears that the
increased number of human bcl-2 copies carried by this
transgenic line influences both the frequency of prostatic
hypertrophy and the severity of this phenotype. Since
human bcl-2 expression was found to be limited to the
epithelial cells in these mice, this phenotype is enigmatic
but extremely interesting, because it involves increased
stromal as well as epithelial cell content of the gland. The
protein encoded by the bcl-2 gene is known to suppress
apoptosis, and it is reasonable to speculate that the overexpression of bcl-2 in the epithelial cells of transgenic
mouse prostate glands is increasing their life span, resulting in abnormal accumulation of epithelial cells as
the mouse ages. However, we have not found any evidence that the human bcl-2 gene is expressed in the stromal cells of these transgenic mouse prostate glands, and
so the cause of the accumulation of stromal cells in these
same tissues is likely not due to the direct action of bcl-2
protein. In the future, examination of these mice might
provide evidence for the production of excess stromal
cell growth factors by the aberrantly long-lived prostatic
epithelium [25]. Likewise, this development of a benign
hypertrophic condition in the prostate glands of some of
these transgenic mice provides the basis for further
analysis as to whether bcl-2 might be an important factor
in the development of human BPH. A preliminary study
Abnormal Prostates in Transgenic Mice
25
Fig. 6. Immunohistochemical staining of ventral prostate tissue obtained from a first-generation heterozygote male from line 5 shows
localization of the human bcl-2 protein to the epithelial cells. The anti-bcl-2 antibody used in this immunostaining protocol does not stain
sections of control (nontransgenic) mouse prostate glands. ×200.
Fig. 7. A: Microscopic analysis of thin sections made from ventral prostate glands obtained from control mouse prostate gland. B:
Severely affected male mouse (3 months old) from line 5 shows phenotypic evidence for cellular accumulation in this specimen. ×100.
already suggests that bcl-2 expression might be elevated
in epithelial cells of human BPH tissue [26].
In summary, these transgenic mice provide a novel
animal model for studying neoplastic development of
the prostate, with particular emphasis on the role of
apoptosis regulation in such development. At this
time, all the progeny mice that we have analyzed have
been heterozygotes. It is possible that homozygotic
progeny will have increased production of human
bcl-2 protein in the prostate, and therefore will develop a more pronounced and consistent abnormal
prostate phenotype. Likewise, these animals will be
26
Zhang et al.
followed through their aging process to determine
whether increased bcl-2 production might be a factor
in the subsequent development of prostate cancers.
ACKNOWLEDGMENTS
This work was supported by grants from the National Institutes of Health (CA48089), the CaPCure
Foundation, the David Koch Foundation, the New
York Academy of Medicine, and the ColumbiaPresbyterian Medical Center Urological Research
Fund.
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