[CANCER RESEARCH 60, 5125–5133, September 15, 2000]
Chemoprevention of Prostate Carcinogenesis by ␣-Difluoromethylornithine in
TRAMP Mice1
Sanjay Gupta, Nihal Ahmad, Susan R. Marengo, Gregory T. MacLennan, Norman M. Greenberg, and
Hasan Mukhtar2
Departments of Dermatology [S. G., N. A., H. M.], Urology [S. R. M.], and Pathology [G. T. M.], Case Western Reserve University, Cleveland, Ohio 44106, and Department of
Cell Biology, Baylor College of Medicine, Houston, Texas 77030 [N. M. G.]
ABSTRACT
INTRODUCTION
Development of effective chemopreventive agents for human consumption requires conclusive evidence of their efficacy in animal models that
have relevance to human diseases. Transgenic adenocarcinoma mouse
prostate (TRAMP) is an excellent model of prostate cancer that mimics
progressive forms of human disease inasmuch as 100% of males develop
histological PIN by 8 –12 weeks of age that progress to adenocarcinoma
with distant site metastases by 24 –28 weeks of age. In these animals,
ornithine decarboxylase (ODC) activity (>3-fold) as well as protein expression (>4-fold) was found to be markedly higher in the dorsolateral
prostate as compared with the nontransgenic littermates, suggesting their
suitability to determine the chemopreventive effect of ␣-difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ODC,
against prostate cancer. Using male TRAMP mice, we studied the effect of
oral consumption of DFMO on development of prostate carcinogenesis
and surrogate end point biomarkers related to prostate cancer progression. In two independent experiments, each consisting of 8 animals on test,
the cumulative incidence of prostatic cancer development at 28 weeks of
age in 16 untreated TRAMP mice was 100% (16 of 16), whereas 94% (15
of 16) and 69% (11 of 16) of the animals exhibited distant site metastases
to lymph nodes and lungs, respectively. Oral consumption of 1% DFMO
(w/v) in the drinking water to TRAMP mice from 8 to 28 weeks of age
resulted in a significant decrease in (a) weight (59%) and volume (66%) of
prostate, (b) genitourinary weight (63%), and (c) ODC enzyme activity
(52%) in the dorsolateral prostate. Importantly, in none of the DFMO-fed
TRAMP mice were any distant metastases to lymph node and lungs
observed. Furthermore, DFMO treatment resulted in the marked reduction in the protein expression of proliferation cell nuclear antigen, ODC,
and probasin in the dorsolateral prostate. The protein expression of
antimetastases markers, i.e., E-cadherin and ␣- and ␤-catenin, was found
to be restored in DFMO-fed animals as compared with the non-DFMOfed mice. These chemopreventive effects of DFMO were further confirmed
by immunohistochemical analysis of the dorsolateral prostate. Histological analysis of the dorsolateral prostate of DFMO-fed animals displayed
marginal epithelial stratification, a small number of cribriform structures,
elongated hyperchromatic epithelial nuclei, and a significant increase in
apoptotic index. Non-DFMO-fed animals, on the other hand, displayed
extensive epithelial stratification with profound cribriform structures accompanied with marked thickening, remodeling, and hypercellularity of
the fibromuscular stroma. In nontransgenic littermates fed with DFMO,
no significant alterations in the above parameters were evident. These
data demonstrate that ODC represents a promising and rational target for
chemoprevention of human prostate cancer and that TRAMP mice are
excellent models for screening of novel drugs and chemopreventive regimens for potential human use.
CaP3 is the most commonly diagnosed solid tumor and the second
leading cause of cancer mortality in American men (1). According to
an estimate by the American Cancer Society, ⬃18% of the male
population in the United States will develop invasive CaP during their
lifetime (1, 2). The development of CaP in humans has been viewed
as a multistage process, involving the onset as small latent carcinoma
of low histological grade to large metastatic lesion of higher grade (3).
Unfortunately, there are limited treatment options available for this
disease because chemotherapy and radiation therapy are largely ineffective, and metastatic disease frequently develops even after potentially curative surgery (4 – 6). Chemoprevention could be an effective
approach to reduce CaP incidence (7, 8). Indeed, CaP is an excellent
candidate disease for chemoprevention because of its diagnosis in
elderly men, and therefore even a modest delay in the neoplastic
development achieved through pharmacological or nutritional intervention could result in a substantial reduction in the incidence of this
clinically detectable disease (9).
For relevance to human population, chemoprevention studies
should be conducted in animal models that mimic progressive forms
of human disease and possess surrogate end point biomarkers for
rapid screening of drugs (10 –12). In recent years, genetically manipulated animal models have emerged as resources for developing
strategies against many pathological conditions, including cancer (13–
15). Advantages of these genetically manipulated animals for chemoprevention studies include induction of carcinogenesis by discrete
genetic changes and the ability to modulate oncogenes or tumor
suppressor genes implicated in human cancers (16 –18). One strength
of transgenic models is that in these animals, cancer arises from
normal cells in their natural tissue microenvironment and progress
through multiple stages, as does human cancer (19).
For CaP very few genetically manipulated models are available (20,
21). One such model is TRAMP (22). TRAMP mice express a PB-Tag
transgene consisting of the minimal ⫺426/⫹28-bp regulatory element
of the rat probasin promoter directing prostate-specific epithelial
expression of the SV40 early genes (T/t antigens; Refs. 22 and 23).
TRAMP is an excellent model that serves as a general prototype of the
pathways, parameters, and molecular mechanisms of multistage prostate tumorigenesis (22). TRAMP males develop spontaneous multistage prostate carcinogenesis that exhibits both histological and molecular features similar to that of human CaP (24). TRAMP males
characteristically express the PB-Tag transgene by 8 weeks of age and
develop distinct pathology in the epithelium of the dorsolateral prostate by 10 weeks of age. Distant site metastasis can be detected as
early as 12 weeks of age in male TRAMP mice, and by 28 weeks of
age, 100% of the animals harbor CaP that metastasizes to the lymph
nodes and lungs (22, 24).
In the present study, we determined the use of TRAMP mice for
Received 2/14/00; accepted 7/10/00.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported by United States Public Health Service Grants CA RO1 78809 (to H. M.),
CA58204 and CA84296 (to N. M. G.), by funds from American Institute for Cancer
Research (to H. M.), Department of Defense DAMD 17-00-1-0527 (to H. M.), and Cancer
Research Foundation of America (to S. G. and N. A.).
2
To whom requests for reprints should be addressed, at Department of Dermatology,
Case Western Reserve University, 11100 Euclid Avenue, Cleveland, Ohio 44106. Phone:
(216) 368-1127; Fax: (216) 368-0212; E-mail: [email protected].
3
The abbreviations used are: CaP, prostate cancer; DFMO, ␣-difluoromethylornithine;
ODC, ornithine decarboxylase; mPB-1, murine probasin-1; PCNA, proliferating cell
nuclear antigen; HRP, horseradish peroxidase; PIN, prostatic intraepithelial neoplasia;
GU, genitourinary.
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PROSTATE CANCER CHEMOPREVENTION BY DFMO IN TRAMP MICE
CaP chemoprevention studies. We used DFMO as a chemopreventive
agent to investigate its efficacy for CaP. Our choice of DFMO is based
on the fact that it is an irreversible “suicide inhibitor” of the enzyme
ODC, and ODC is being increasingly appreciated as a target for CaP
chemoprevention because of the following reasons: (a) ODC is shown
to play a role during CaP development in the murine prostate carcinogenesis model (25–27), (b) ODC is overexpressed in prostate tissue
and prostatic fluid in human subjects with CaP (28), and (c) our
studies have shown that ODC enzyme activity and protein expression
are elevated in the dorsolateral prostate of TRAMP mice. The results
from this study suggested that ODC is a rational target and end point
biomarker for CaP development and progression, and TRAMP is an
excellent model for screening for novel drugs and chemopreventive
agents against CaP.
MATERIALS AND METHODS
regrowth, in another set of experiment, 12 male TRAMP mice of 8 weeks of
age were divided into two groups. The control group was fed with 1% DFMO
(w/v) for 20 weeks, whereas the experimental group was fed with 1% DFMO
(w/v) for 14 weeks and later returned to regular drinking water for 6 weeks,
was sacrificed, and was studied for prostate tumorigenesis as described earlier.
Immunoblotting. The dorsolateral prostate was removed from the transgenic and nontransgenic littermates, homogenized in lysis buffer [50 mM
Tris-HCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 20 mM NaF, 100 mM
Na3VO4, 0.5% NP40, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride,
10 ␮g/ml aprotinin, and 10 ␮g/ml leupeptin (pH 7.4)] at 4°C to prepare cell
lysates. Appropriate amount of protein (25–50 ␮g) was resolved over 8 –14%
Tris-Glycine polyacrylamide gel and then transferred onto the nitrocellulose
membrane. The blots were blocked using 5% nonfat dry milk and probed using
appropriate primary antibody of ODC (Neomarkers Inc., Union City, CA),
mPB-1 (from N. M. G.’s laboratory), E-cadherin, PCNA, and ␣- and ␤-catenin
(Santa Cruz Biotechnology, Santa Cruz, CA) in blocking buffer overnight at
4°C. The membrane was then incubated with antimouse or antirabbit secondary antibody HRP conjugate (Amersham Life Sciences Inc., Arlington Heights,
IL) followed by detection using the enhanced chemiluminescence kit (Amersham Life Sciences Inc., Arlington Heights, IL). Equal loading of protein was
confirmed by stripping the membrane and reprobing it with ␤-actin primary
antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and appropriate secondary HRP conjugate.
Tissue Processing and Histopathology. The dorsolateral prostate was
excised and fixed overnight in (10%) zinc-buffered formalin and then transferred to 70% ethanol. Sections (4 ␮m) were cut from paraffin-embedded
tissue and mounted on slides. The sections were stained with H&E as described
previously (24). Histological sections were reviewed by light microscopy for
the presence of CaP and classified as PIN (epithelial stratification with occasional mitotic figures or cribriform pattern); well- (multiple epithelial mitotic
figures and apoptotic bodies, invasive glands with stromal hypercellularity),
moderately (many acini completely filled with tumor yet still forming some
glandular structures), or poorly (sheets of malignant cells with little or no
glandular formation) differentiated CaP; or atrophic glands only (no identifiable tumor deposits).
Immunohistochemical Analysis. Sections (4 ␮m) were cut from paraffinembedded tissues. Immunostaining was performed using antibody for mPB-1,
PCNA, E-cadherin, and ␣- and ␤-catenin with appropriate dilutions and was
replaced with either normal host serum or block for negative controls, followed
by counterstaining with either a weak hematoxylin stain or methyl green as
described previously (22, 24). The stained slides were visualized on a ZeissAxiophot DM HT microscope (Zeiss-Axiophot, Germany). Images were captured with an attached camera linked to a computer.
Metastasis Examination. The india ink method was used to examine
cancer metastasis to lungs. For this, the animals were sacrificed and the
respiratory system was excised. India ink was injected through the trachea into
the lungs using a 5-ml syringe with a fine tip until the lungs were completely
filled with ink. The trachea was then tied with a thread. The ink absorbs in
whole tissue, with the exception of places where the metastasis had occurred.
Thus, the metastatic tissues were visible as unstained spots in lungs, which
were stained blue.
Scoring of Apoptotic Cells. Apoptotic cells were evaluated by the morphological examination of H&E-stained sections under light microscopy. Scoring was done under the microscope using the Optimas 6 software program
(Optimas Corp., Bothell, WA). Apoptotic index (%) was calculated by dividing the number of apoptotic cells by the total number of cells counted per
cross-section of a sample of the prostate.
Densitometry and Statistical Analysis. Densitometric measurements of
the bands in immunoblot analysis were performed using the Scion Image
computer program (Scion Corp., Frederick, MD). The data are expressed as
mean ⫾ SE. The significance between the control and experimental groups
was obtained by using the Student’s t test, and a P ⱕ 0.05 was taken as
significant in all of the experiments.
Animals. The male and female heterozygous TRAMP mice, line PB-Tag
8247, were developed in the laboratory of one of us (N. M. G.) at Baylor
College of Medicine (Houston, Texas) and were bred and maintained at
Association for Assessment and Accreditation of Laboratory Animal Care
(AAALAC) internationally accredited Animal Resource Facility of Case Western Reserve University. All of the experiments were conducted using the
highest standards for animal care in accordance with the NIH Guidelines for
the Care and Use of Laboratory Animals. Transgenic males for these studies
were routinely obtained as [TRAMP ⫻ C57BL/6]F1 or as [TRAMP ⫻ C57BL/
6]F2 offspring. Identity of transgenic mice was established by the PCR-based
DNA screening as described earlier (29).
ODC Enzyme Activity. Male TRAMP mice (8, 16, and 24 weeks old) and
their nontransgenic littermates of the same age were used for initial studies.
The animals had access to food and water ad libitum, and in each group, six
animals were used. The animals from both transgenic and nontransgenic
groups were sacrificed by cervical dislocation, and dorsolateral prostate was
removed. The tissue was homogenized at 4°C in a glass-to-glass homogenizer
in 10 volumes of ODC buffer [50 mM Tris-HCl buffer (pH 7.5) containing 0.1
mM EDTA, 0.1 mM DTT, 0.1 mM pyridoxal-5-phosphate, 1 mM 2-mercaptoethanol and 0.1% Tween-80]. The homogenate was centrifuged at 14,000 ⫻ g
at 4°C, and the supernatant was used for enzyme determination. ODC activity
was determined by measuring the release of 14CO2 from DL-[1-14C]ornithine
hydrochloride, as described earlier (28). Briefly, 100 ␮l of the supernatant
were added to 0.25 ml of the assay mixture [35 mM sodium phosphate (pH 7.2),
0.2 mM pyridoxal phosphate, 4 mM DTT, 1 mM EDTA, 0.4 mM L-ornithine
containing 0.5 ␮Ci of DL-[1-14C]ornithine hydrochloride] in a 15-ml corex
centrifuge tube equipped with rubber stoppers and central well assemblies
containing 0.2 ml of ethanolamine and methoxyethanol in a 2:1 (v/v) ratio.
After incubation at 37°C for 60 min, the reaction was terminated by the
addition of 0.5 ml of 2 M citric acid using a 21-gauge needle/syringe.
The incubation was continued for 1 h. Finally, the central well containing the
ethanolamine:methoxyethanol mixture to which 14CO2 has been trapped was
transferred to a vial containing 10 ml of toluene-based scintillation fluid and 2
ml of ethanol. The radioactivity was measured in a Beckman LS 6000 SC
liquid scintillation counter.
Study Design for DFMO Chemoprevention. The effect of 1% DFMO
(w/v) consumption on prostate carcinogenesis in TRAMP mice was studied in
two independent experiments. Throughout the experiment all of the animals
had ad libitum access to laboratory chow. In each experiment, 16 male
TRAMP mice of 8 weeks of age were divided in two groups. The experimental
group was provided with 1% DFMO (ILEX Oncology, San Antonio, TX)
solution (w/v) in drinking water ad libitum for 20 weeks. The second group of
untreated mice received drinking water ad libitum and served as the control in
the chemoprevention trial. Based on average consumption of 4.6 ml of water/
mouse/day, each mouse consumed ⬃50 mg of DFMO/day. This feeding
regimen of DFMO was well tolerated by TRAMP as well as by nontransgenic RESULTS
mice, a finding consistent with the animals in carcinogenesis and biochemical
ODC Enzyme Activity and Protein Expression in TRAMP
studies conducted in different laboratories (16, 30 –32). Additional untreated
Mice. ODC, the rate-limiting enzyme in polyamine biosynthesis
and treated nontransgenic littermates were also included in the study.
To investigate the effect of cessation of DFMO feeding on prostate tumor pathway, is associated with growth and development of neoplastic
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tissue and is found to be up-regulated in almost all epithelial cancers,
including CaP (33–35). In this experiment, we first compared the
levels of ODC enzyme activity and protein expression in dorsolateral
prostate of TRAMP mice and their nontransgenic littermates at 8
weeks of age. As shown in Fig. 1A, basal levels of ODC enzyme
activity were similar in nontransgenic and TRAMP mice. ODC activity in 16- and 24-week-old TRAMP mice was 2.5- and 3.3-fold
higher than in age-matched nontransgenic littermates. The ODC protein expression was significantly higher in 16- and 24-week-old
TRAMP mice (4.4-fold and 5.6-fold, respectively) compared with the
nontransgenic littermates (Fig. 1B). This observation offers the unique
opportunity to use the TRAMP model for DFMO chemoprevention
studies.
Effect of DFMO Consumption on Prostate Tumorigenesis in
TRAMP Mice. DFMO feeding for 20 weeks did not significantly
affect the body weight gain profile of nontransgenic littermates comFig. 2. Effect of DFMO consumption on the weight gain profile of TRAMP mice and
their nontransgenic littermates. The weight gain profiles of nontransgenic (control),
nontransgenic (DFMO-fed), transgenic (control), and transgenic (DFMO-fed) male mice
from 8 –28 weeks are shown. ⴱ, P ⬍ 0.05, Student’s t test, TRAMP (control) versus
TRAMP (DFMO-fed). Bars, SE of eight mice.
Fig. 1. ODC enzyme activity and protein expression in the dorsolateral prostate of male
TRAMP mice and their nontransgenic littermates. A, ODC enzyme activity was measured
at 8, 16, and 24 weeks of age. A marked difference in ODC enzyme activity was observed
at 16 and 24 weeks of age. Data represents mean ⫾ SE of six mice. B, ODC protein
expression in TRAMP mice and their nontransgenic littermates at 16 and 24 weeks of age.
Data from a representative experiment is shown here. Densitometric analysis of the bands
in TRAMP mice and their nontransgenic littermates at 16 and 24 weeks of age indicates
a marked difference in ODC protein expression. ⴱ, P ⬍ 0.001, Student’s t test, TRAMP
versus nontransgenic mice. Bars, SE of six mice.
pared with their corresponding controls. In contrast, TRAMP mice fed
with DFMO were 8 g lighter, 28 ⫾ 3 versus 36 ⫾ 4, than the
non-DFMO-fed TRAMP mice. The difference in the body weight
appeared to be attributable to the hyperproliferation of the GU apparatus and local abdomen region in the non-DFMO-fed TRAMP mice
(Fig. 2).
To investigate the effects of DFMO consumption on CaP in
TRAMP mice, in two independent experiments, DFMO was fed for
20 weeks starting at 8 weeks of age to TRAMP mice and their
nontransgenic littermates. As summarized in Table 1, in the first
experiment, eight of eight TRAMP mice in the non-DFMO-fed group
developed severe CaP with remarkable local invasiveness, and distant
site metastases to lymph (seven of eight) and lungs (six of eight) were
also observed. In contrast, none of the eight DFMO-fed TRAMP mice
on the test developed metastases to lymph nodes or lungs. However,
in all of the animals, mild local invasiveness was observed in the
seminal vesicles and local prostate region. Similarly, in the repeat
experiment, all eight mice in the non-DFMO-fed group developed
fully malignant invasive CaP with metastases to lymph (eight of eight)
and lungs (five of eight). At the termination of the experiment, at 28
weeks in the DFMO-fed group, none of the eight mice exhibited any
metastases to lymph nodes and lungs.
We next investigated the effect of DFMO consumption on the GU
apparatus in TRAMP mice. As observed visibly, DFMO feeding
resulted in almost none to complete absence of hyperplasia in the GU
region, especially of the seminal vesicles. The most striking features
of DFMO consumption were evident by a significant decrease in
prostate weight (59%) and volume (66%) as compared with the
non-DFMO-fed TRAMP mice. A significant reduction in GU weight
(63%) in the DFMO-fed TRAMP mice was observed, compared with
the non-DFMO-fed TRAMP mice. No significant changes were observed in the DFMO-fed nontransgenic littermates as compared with
their corresponding control (Table 1).
Effect of DFMO Consumption on Histological Features in Prostate of TRAMP Mice. As shown in Fig. 3A, a typical dorsolateral
prostate from a control, nontransgenic littermate is composed of acini
with abundant eosinophilic intralumenal secretions. The acini are
lined by a layer of well-organized columnar secretory epithelium
possessing round and inconspicuous nuclei. A single layer of thin, flat
basal epithelial cells with elongated nuclei typically surrounds the
columnar epithelium, and a fibromuscular stroma containing three to
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PROSTATE CANCER CHEMOPREVENTION BY DFMO IN TRAMP MICE
Table 1 Effect of DFMO on the morphology of prostate and GU weight in TRAMP mice and their nontransgenic littermatesa
Animals with metastasisc
Groupb
Experiment 1
Nontransgenic water-fed
Nontransgenic DFMO-fed
Transgenic water-fed
Transgenic DFMO-fed
Experiment 2
Nontransgenic water-fed
Nontransgenic DFMO-fed
Transgenic water-fed
Transgenic DFMO-fed
Cumulative
Nontransgenic water-fed
Nontransgenic DFMO-fed
Transgenic water-fed
Transgenic DFMO-fed
No. of
Animals
Prostate
cancer
Lymph
Prostate
weight (mg)
Prostate
volume (mm3)
GU weight (g)
8
8
8
8
None
None
Severe
Mild
0/8 (0%)
0/8 (0%)
7/8 (87.5%)
0/8 (0%)
0/8 (0%)
0/8 (0%)
6/8 (75%)
0/8 (0%)
12.9 ⫾ 1.1
11.4 ⫾ 1.4
63.2 ⫾ 8.4
24.6 ⫾ 2.4d
8.47 ⫾ 0.53
7.62 ⫾ 0.49
46.92 ⫾ 9.12
16.36 ⫾ 1.54d
0.38 ⫾ 0.05
0.34 ⫾ 0.03
2.69 ⫾ 0.48
0.94 ⫾ 0.11d
8
8
8
8
None
None
Severe
Mild
0/8 (0%)
0/8 (0%)
8/8 (100%)
0/8 (0%)
0/8 (0%)
0/8 (0%)
5/8 (62.5%)
0/8 (0%)
11.6 ⫾ 1.0
10.8 ⫾ 1.2
76.4 ⫾ 10.2
30.5 ⫾ 3.6d
7.87 ⫾ 0.48
7.16 ⫾ 0.44
54.64 ⫾ 9.68
18.22 ⫾ 2.08d
0.32 ⫾ 0.04
0.29 ⫾ 0.04
3.12 ⫾ 0.52
1.16 ⫾ 0.12d
16
16
16
16
None
None
Severe
Mild
0/8 (0%)
0/8 (0%)
15/16 (93.7%)
0/16 (0%)
0/8 (0%)
0/8 (0%)
11/16 (68.7%)
0/16 (0%)
12.2 ⫾ 1.2
11.2 ⫾ 1.5
67.8 ⫾ 9.8
27.5 ⫾ 3.2d
8.14 ⫾ 0.52
7.38 ⫾ 0.50
50.76 ⫾ 9.46
17.28 ⫾ 1.88d
0.35 ⫾ 0.05
0.31 ⫾ 0.04
2.90 ⫾ 0.50
1.06 ⫾ 0.12d
Lungs
The data represented in two independent experiments is the mean ⫾ SE of eight mice.
Mice (8 weeks of age) were given plain drinking water (for the control group) or DFMO in drinking water for 20 weeks. At the age of 28 weeks, the animals were sacrificed and
studied for prostate tumorigenesis and metastasis.
c
Metastasis in the lymph was examined microscopically, whereas metastasis in lungs was examined by india ink method as described in “Materials and Methods.”
d
P ⬍ 0.001, water-fed control TRAMP compared with DFMO-treated TRAMP mice, Student’s t test.
a
b
four cell layers of stratified smooth muscle surrounds the acinus. The
dorsolateral prostate of the nontransgenic littermate supplemented
with DFMO for 20 weeks was entirely similar to that of the nontransgenic water-fed group (Fig. 3A).
A detailed histopathological investigation of neoplastic progression
of the dorsolateral prostate of non-DFMO-fed TRAMP mice at 28
weeks of age displayed poorly differentiated PIN characterized by
profound cribriform structures and numerous apoptotic bodies, invasive glands accompanied by marked thickening, remodeling, and
hypercellularity of the fibromuscular stroma (Fig. 3A). In sharp contrast, the experimental group of DFMO-fed TRAMP mice exhibited
well-differentiated epithelial stratification with a marked reduction in
cribriform structures, thickening, and remodeling of the fibromuscular
stroma underlying the abnormal prostatic epithelium along with little
or no glandular formation (Fig. 3A). Compared with the non-DFMO
fed TRAMP mouse, the prostate of DFMO-fed mouse exhibits acini
with many epithelial nuclei, which are elongated and hyperchromatic,
with clumping of the chromatin and an increased nuclear:cytoplasmic
ratio. DFMO feeding also resulted in a significant increase in the
number of apoptotic cells, which occurred sporadically and are darkly
stained (Fig. 3B). A high apoptotic index was observed in the DFMOfed group, compared with non-DFMO-fed TRAMP mice (Fig. 3C).
Effect of DFMO Consumption on ODC Activity and Protein
Expression in TRAMP Mice. TRAMP males exhibit high basal
levels of ODC enzyme activity and protein expression in the dorsolateral prostate, which is associated with cell invasion and regarded as
a marker of cell proliferation (16, 25, 28, 30). As shown in Fig. 4A,
DFMO feeding for 20 weeks to TRAMP mice (ODC activity,
218.9 ⫾ 15.3 pmol/h/mg protein) resulted in a significant decrease
(P ⬍ 0.001) in ODC enzyme activity in the dorsolateral prostate as
compared with the non-DFMO-fed TRAMP mice (ODC activity,
454.4 ⫾ 31.8 pmol/h/mg protein). Importantly, TRAMP mice fed
with DFMO for 20 weeks also showed a significant reduction in ODC
protein expression in the dorsolateral prostate as compared with the
non-DFMO-fed TRAMP group (Fig. 4B). The densitometric analysis
of the bands (normalized for ␤-actin) showed a significant decrease
(P ⬍ 0.001) in ODC protein expression (54%) in DFMO-fed TRAMP
mice compared with the non-DFMO-fed TRAMP mice (Fig. 4C).
DFMO supplementation to the nontransgenic littermates did not cause
any significant alterations in the ODC activity and protein expression
in the dorsolateral prostate (Fig. 4).
Effect of DFMO Consumption on Proliferation Marker(s) in
TRAMP Mice. TRAMP mice reproducibly express the PB-Tag
transgene in a male-specific and developmentally regulated fashion.
Probasin (PB) is an androgen responsive gene expressed specifically
in prostate epithelial cells and has been shown to be up-regulated in
CaP (34 –36). Therefore, we next determined the effect of DFMO
consumption on the protein expression of probasin in the dorsolateral
prostate of these mice. TRAMP mice fed with DFMO for 20 weeks
exhibited significant inhibition in probasin protein expression in the
dorsolateral prostate (Fig. 5A). These results were further confirmed
by comparing the relative densities of the bands where 49% reduction
(P ⬍ 0.001) was observed in the DFMO-fed TRAMP mice as compared with the non-DFMO-fed TRAMP animals (Fig. 5B). The immunohistochemical staining of the dorsolateral prostate in DFMO-fed
TRAMP mice exhibited a significant decrease in probasin protein
expression as compared with the non-DFMO-fed TRAMP group (Fig.
6). However, in the nontransgenic littermates, DFMO supplementation was not found to result in noticeable alterations in the probasin
expression compared with their corresponding control (Figs. 5 and 6).
Because DFMO is a known antiproliferative agent (37, 38), we
investigated the effect of DFMO chemoprevention on the ubiquitous
and molecular marker for increased proliferation, PCNA. PCNA
serves as a requisite auxiliary protein for DNA polymerase ␦-driven
DNA synthesis and is cell cycle regulated (39). DFMO consumption
for 20 weeks resulted in a marked reduction in PCNA protein expression in the dorsolateral prostate of TRAMP mice compared with the
non-DFMO-fed TRAMP mice (Fig. 5A). The densitometric analysis
of the bands exhibited a 32% decrease (P ⬍ 0.001) in PCNA protein
expression in the DFMO-fed TRAMP mice compared with the nonDFMO-fed TRAMP group (Fig. 5B). These results were further
confirmed by immunohistochemical analysis (Fig. 6). Feeding of
DFMO to nontransgenic littermates did not result in any significant
alterations in PCNA protein expression (Figs. 5 and 6).
Effect of DFMO Consumption on Metastases Marker(s) in
TRAMP Mice. Uncontrolled progression of CaP leads to distant site
metastases, which is responsible for the majority of CaP-related
deaths in humans (40, 41). Therefore, we evaluated the effect of
DFMO on the markers of CaP metastasis. The progression of CaP
occurs via loss of functional expression of E-cadherin (42, 43), and
the cadherin-catenin complex suppression and their loss from the
epithelial cell have been described as increased invasiveness and
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fed TRAMP mice exhibited a marked restoration of E-cadherin in the
basement membrane compared with the non-DFMO-fed TRAMP
mice (Fig. 6). No significant alteration in the E-cadherin protein
expression was observed in the DFMO-fed nontransgenic littermates
when compared with the corresponding control (Figs. 5 and 6).
We next analyzed the effect of DFMO consumption on protein
expression of ␣- and ␤-catenin in TRAMP mice. DFMO feeding for
20 weeks was effective in restoring the protein expression of ␣-catenin as well as ␤-catenin in the dorsolateral prostate of TRAMP mice
compared with the non-DFMO-fed TRAMP mice (Fig. 5A). The
densitometric analysis of the immunoblot showed that DFMO feeding
results in a significant restoration (62%; P ⬍ 0.001) of the protein
levels of ␣- and ␤-catenin (537%; P ⬍ 0.001; Fig. 5B). These results
were further confirmed by immunohistochemical analysis where
DFMO feeding to TRAMP mice resulted in the restoration of ␣- and
␤-catenin compared with the non-DFMO-fed TRAMP mice (Fig. 6).
In the nontransgenic littermates, however, DFMO supplementation
was not found to result in noticeable alterations in the protein expression of these markers (Figs. 5 and 6).
Effect of DFMO Cessation on Prostate Tumor Regrowth in
TRAMP Mice. The experiment was conducted to investigate
whether the cessation of DFMO feeding causes a regrowth of CaP in
TRAMP mice. As shown in Table 2, 20 weeks of DFMO feeding was
effective in reducing CaP growth and invasion in TRAMP mice. The
experimental animals, which received regular drinking water for 6
Fig. 3. Histological examination of dorsolateral prostate in male TRAMP mice and
their nontransgenic littermate after DFMO feeding. A, tissue sections were stained with
H&E and examined at 40⫻ magnification. A typical dorsolateral prostate from a nontransgenic mouse exhibited acini with abundant eosinophilic intralumenal secretions.
DFMO feeding to a nontransgenic mouse did not exhibit any noticeable alterations. In
TRAMP mice (control), extensive epithelial stratification with profound cribriform structures accompanied with marked thickening, remodeling, and hypercellularity of the
fibromuscular stroma was observed. DFMO feeding to TRAMP mice resulted in a marked
reduction in epithelial stratification and cribriform structures. B, a detailed examination of
a DFMO-fed section (80⫻) reveals acini with many elongated hyperchromatic epithelial
nuclei, with clumping of the chromatin and an increased nuclear:cytoplasmic ratio
compared with the transgenic control at a similar magnification. Apoptotic cells were
ascertained by morphological criteria of the cells examined under light microscopy. The
details are described in the “Materials and Methods” section. Arrows, the apoptotic cells.
C, a high apoptotic index was observed in the DFMO-fed group, which was exhibited by
a significant increase in the number of apoptotic bodies and dense staining of the nuclei,
compared with non-DFMO-fed TRAMP mice. ⴱ, P ⬍ 0.001, Student’s t test, TRAMP
(control) versus TRAMP (DFMO-fed). Bars, SE.
development of metastatic growth to different organs (44). Earlier
studies have shown that the normal pattern of these markers is lost
during CaP progression in TRAMP mice in a fashion that parallels
human CaP (22, 24). DFMO feeding to TRAMP mice for 20 weeks
was effective in a significant restoration of the protein expression of
E-cadherin in the dorsolateral prostate (Fig. 5A). These results were
further confirmed by the densitometric analysis of the immunoblot
where a significant restoration of 246% (P ⬍ 0.001) of E-cadherin
protein was observed in the DFMO-fed TRAMP mice (Fig. 5B). The
immunohistochemical analysis of the dorsolateral prostate in DFMO-
Fig. 4. Effect of DFMO consumption on ODC enzyme activity and protein expression
in TRAMP mice and their nontransgenic littermates. A, ODC enzyme activity in the
dorsolateral prostate of nontransgenic (control), nontransgenic (DFMO-fed), TRAMP
(control), and TRAMP (DFMO-fed) male mice from 8 –28 weeks. A marked decrease in
ODC enzyme activity was observed after DFMO feeding. Data from eight mice per group
are shown here. B, ODC protein expression in the dorsolateral prostate of different
nontransgenic and TRAMP groups. Representative data from four mice per group are
shown here. C, densitometric analysis of ODC protein expression from the above blot
indicates a marked decrease in protein expression after DFMO feeding. ⴱ, P ⬍ 0.001,
Student’s t test, TRAMP (control) versus TRAMP (DFMO-fed). Bars, SE of eight mice.
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PROSTATE CANCER CHEMOPREVENTION BY DFMO IN TRAMP MICE
Fig. 5. Effect of DFMO consumption on the protein expression of proliferation and metastases markers in TRAMP mice and their nontransgenic littermates. A, protein expression
of mPB-1, PCNA, E-cadherin, and ␣- and ␤-catenin in the dorsolateral prostate of nontransgenic (control), nontransgenic (DFMO-fed), TRAMP (control), and TRAMP (DFMO-fed)
male mice from 8 –28 weeks. A marked decrease in mPB-1 and PCNA and a significant restoration of E-cadherin and ␣- and ␤-catenin protein expression were observed after DFMO
feeding. The control as well as DFMO-fed nontransgenic animals did not exhibit any significant alterations in the protein expression described above. Representative data from four
mice per group are shown here. B, densitometric analysis of the blot was performed, and the results are expressed in relative density where the nontransgenic water-fed group was taken
as the control (100%). Equal loading of protein in the lanes was confirmed by stripping the membrane and reprobing it with ␤-actin primary antibody with appropriate secondary HRP
conjugate. The details are described in the “Materials and Methods” section.
weeks after 14 weeks of DFMO feeding, exhibit moderate local
invasiveness in seminal vesicles and in the local prostate region.
However, no distant site metastasis to lymph nodes and lungs was
observed. The weight and volume of prostate and the GU apparatus
was found to significantly increase (P ⬍ 0.05) in the experimental
group, compared with DFMO-fed TRAMP mice. The histopathological examination of the dorsolateral prostate of the experimental group
exhibit moderately differentiated prostate tumor where acini are completely filled with tumor yet still forming some glandular structures
(data not shown).
DISCUSSION
The present investigation demonstrates the use of TRAMP mouse
for CaP chemoprevention studies and shows that intervention with
DFMO holds promise for use against CaP. For establishing proof of
the principle of biomarkers assessing human CaP risk and for the
translation of knowledge to humans, the chemopreventive potential of
an agent should be validated in the animal models, which emulate
progressive forms of human disease and where the cancer is developed without the need to administer unrealistic large doses of chemical carcinogens or hormones. In recent years, genetically manipulated
animal models are emerging as resources for developing strategies
against many pathological conditions, including cancer (13–15, 17,
18). For CaP, one such model is TRAMP where specific genes are
involved in complex multifactorial process of prostate carcinogenesis.
Furthermore, TRAMP mice that have an intact immune system and
develop spontaneous autochthonous disease provide an opportunity to
evaluate therapeutic modalities (22, 23). TRAMP mice spontaneously
develop CaP through a series of well-defined stages associated with
characteristic alterations in markers of proliferation and metastasis
(24). In this study, we used TRAMP mice to investigate the efficacy
of DFMO against the growth and progression of CaP. Very few
chemoprevention studies have been conducted thus far in transgenic
animal models (16). Because TRAMP mice were found to express
high basal level of ODC enzyme activity and protein expression in the
dorsolateral prostate, we used DFMO as an agent for chemoprevention of CaP.
The other important consideration toward the intervention and
prevention of CaP is the development of surrogate end point marker(s)
that can detect early premalignant changes along the course of tumor
development. It is also desirable that the biomarker should be able to
monitor the efficacy of chemoprevention/treatment. Our previous
study (28) has shown that ODC, the rate-limiting terminal enzyme in
the polyamine biosynthetic pathway, is overexpressed in human CaP
and could serve as a biomarker for early detection of CaP in humans.
Because ODC overexpression and accumulation of polyamines, i.e.,
putrescine, spermine, and spermidine, are common features in most
primary epithelial human cancers, such as cancers of skin (45), breast
(46), and prostate (47), ODC could be exploited as a rational target for
chemoprevention. In the present study, DFMO supplementation
caused a rapid and substantial decrease in ODC enzyme activity and
protein expression in the dorsolateral prostate with a significant decrease in hyperproliferation of the GU apparatus. Further, histopathological as well as immunohistochemical and immunoblotting analyses
for proliferation markers, i.e., PCNA and probasin, showed that
DFMO is an effective chemopreventive agent against CaP. The histopathological analysis was also found to correlate with the biochemical response as well as the proliferation. The other significant finding
of this study is the apoptotic index, which was found to be significantly greater in DFMO-fed TRAMP mice. This result suggests that
in addition to inhibiting proliferation, apoptosis plays a significant
role in the DFMO inhibition of the growth of CaP in TRAMP mice.
These findings are in accordance with recent studies (48, 49) where
DFMO-induced apoptosis is shown. These results support our hypothesis that ODC could be used as a rational target and DFMO as an ideal
agent for chemoprevention in prostate carcinogenesis.
Invasion of CaP to distant sites causes metastatic disease that is
regarded as the major cause of CaP-related deaths in humans (40, 41).
Tumor cells acquire this increased invasive potential by a complex
pathway, which include decreased cell substrate attachment and cellcell adhesion, as well as increased cell motility. In the TRAMP model,
loss of E-cadherin has been suggested to play a major role in modulating metastasis (42, 43). Many studies have shown that the cadherincatenin complex is correlated with the loss of cellular differentiation
and acquisition of invasive and metastatic potential in human tumors,
including head and neck (50, 51), breast (52, 53) bladder (54), gastric
(55), prostate (56), colon (57), and basal cell carcinoma of the skin
(58). We have demonstrated that DFMO feeding to TRAMP mice
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Fig. 6. Immunohistochemical analysis of proliferation and metastases markers in TRAMP mice and their nontransgenic littermates after DFMO feeding. Immunohistochemical
detection of mPB-1, PCNA, E-cadherin, and ␣- and ␤-catenin in the dorsolateral prostate of nontransgenic (control), nontransgenic (DFMO-fed), TRAMP (control), and TRAMP
(DFMO-fed) male mice from 8 –28 weeks. A representative figure from each group is shown here. In TRAMP mice, an extensive mPB-1 staining was observed in the lumen and acinar
regions of the epithelium, whereas extensive PCNA staining was observed in the nuclei. DFMO feeding resulted in the marked reduction in mPB-1 and PCNA staining in these mice.
The control as well as DFMO-fed nontransgenic animals did not exhibit mPB-1 expression, whereas few PCNA stained cells were observed in these groups. Normal E-cadherin and
␣- and ␤-catenin expression was maintained in the basolateral cell-cell borders of the epithelia in nontransgenic control and DFMO-fed animals. In the TRAMP mouse, a significant
loss in the normal expression of E-cadherin and ␣- and ␤-catenin was observed in the dorsolateral prostate, whereas DFMO feeding resulted in a significant restoration of these proteins.
Table 2 Effect of DFMO cessation on the morphology of prostate and GU weight in TRAMP micea
Animals with metastasisc
Group
b
Control (No DFMO)
DFMO feeding for 20 wk
DFMO feeding (14 wk),
followed by water (6 wk)
No. of
Animals
CaP
Lymph
Lungs
Prostate
weight (mg)
Prostate
volume (mm3)
GU weight (g)
6
6
6
Severe
Mild
Moderate
6/6 (100%)
0/6 (0%)
0/6 (0%)
4/6 (66.6%)
0/6 (0%)
0/6 (0%)
68.6 ⫾ 8.86
28.2 ⫾ 2.84
38.8 ⫾ 4.48d
52.44 ⫾ 9.24
17.54 ⫾ 1.66
24.22 ⫾ 2.12d
2.96 ⫾ 0.51
1.02 ⫾ 0.12
1.83 ⫾ 0.26d
The data represent the mean ⫾ SE of six mice.
Mice (8 weeks of age) were given plain drinking water (for control group) or DFMO in drinking water for 20 or 14 weeks. For the 14-week study, the animals were returned to
plain drinking water after 14 weeks. At the age of 28 weeks, the animals were sacrificed and studied for prostate tumorigenesis and metastasis.
c
Metastasis in the lymph was examined microscopically, whereas metastasis in lungs was examined by using the india ink method as described in “Materials and Methods.”
d
P ⬍ 0.05, DFMO-fed (for 20 weeks) TRAMP versus DFMO-fed (for 14 weeks), Student’s t test.
a
b
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caused a significant restoration of the loss of E-cadherin and ␣- and
␤-catenins. We suggested that this might be a reason for the complete
absence of metastases in these mice.
The biology of CaP includes a well-differentiated histopathological and more benign malignant behavior. TRAMP mice exhibit
PB-Tag transgene expression in prostate cell epithelium. This is
where cancers arise from normal cells, in their microtissue environment, and progress through multiple stages, as does human
cancer (22, 24). Our studies with DFMO chemoprevention have
shown a marked decrease in development and progression of CaP
in TRAMP mice. It is important to emphasize that DFMO treatment to TRAMP mice did not alter the expression of the PB-Tag
transgene because it was readily detectable in both DFMO and
non-DFMO-fed groups (data not shown). Further, the expression of
the PB-Tag transgene has been shown to precede the histological
appearances of carcinoma in the TRAMP mice, as in other transgenic models, suggesting that the proliferative response as a consequence of Tag oncoprotein expression is a prerequisite but not
sufficient for neoplastic transformation (24). These observations
support the hypothesis that cells expressing the transgene are in a
preneoplastic state and that other stochastic events are required to
confer proliferation and ultimately a malignant state.
The use of DFMO as a cancer chemopreventive agent has been
strengthened in recent years. Several reports have shown that oral
administration of DFMO to tumor-bearing animals for ⱖ2 weeks
reduces the frequency and development of pulmonary and liver metastasis and concomitantly inhibits the growth of primary tumors
(59 – 62). DFMO was subsequently shown to inhibit carcinogeninduced cancer development in a number of rodent models (59 – 62).
Parallel to these studies, our results demonstrate that DFMO was able
to suppress tumor development and progression in the TRAMP mouse
as long as DFMO was continuously administered. However, when
DFMO feeding was suspended (at 14 weeks), an increased local
invasiveness and CaP growth were observed after 6 weeks in TRAMP
mice, further suggesting that ODC could be used as a target as well as
a biomarker for CaP chemoprevention. DFMO treatment has been
reported to exhibit ototoxicity in humans (63). However, recent clinical cancer chemoprevention trials indicate that low doses of DFMO
can be given for a longer period to cancer patients without any
detectable ototoxicity or other side effects (38, 60, 64). It is important
to emphasize here that the feeding regimen of DFMO used in this
study did not exhibit any toxic symptoms in TRAMP mice or in their
nontransgenic littermates.
In summary, the chemoprevention studies by DFMO in TRAMP
mice were effective, and all of the markers of proliferation examined
nicely correlated with tumor invasiveness that was affected by
DFMO. We conclude that ODC represents a promising and rational
target for CaP chemoprevention, and TRAMP mice are excellent
models for screening of novel drugs and CaP chemopreventive regimens for potential human use.
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Chemoprevention of Prostate Carcinogenesis by α
-Difluoromethylornithine in TRAMP Mice
Sanjay Gupta, Nihal Ahmad, Susan R. Marengo, et al.
Cancer Res 2000;60:5125-5133.
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