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Method for identifying prostate cells in semen using flow cytometry

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The Prostate 36:168–171 (1998)
Dihydrotestosterone Prevents Spontaneous
Adenocarcinomas in the Prostate-Seminal
Vesicle in Aging L-W Rats
Morris Pollard*
Lobund Laboratory, University of Notre Dame, Notre Dame, Indiana
BACKGROUND. Gene-regulated mechanisms govern tumor development, but the actual
development of tumors can be suppressed or promoted by epigenetic factors. Lobund-Wistar
(L-W) rats are genetically predisposed to development of spontaneous and induced metastasizing moderately differentiated adenocarcinomas in the prostate-seminal vesicle (P-SV)
complex. In L-W rats with one slow-release subcutaneous implant of dihydrotestosterone
(DHT) (5a-Androstan-17b-ol-3-one), the development of induced P-SV tumors 14 months
later was significantly suppressed, with involution of testes, aspermia, and absence of detectable serum testosterone. The tumor-suppressive effect of DHT was confirmed. Spontaneous
P-SV tumors developed in 57 of 220 control L-W rats (26%) at an average age 20 months.
METHODS. At age 12 months, 70 L-W rats were administered an implant of 40 mg of DHT,
and 75 untreated rats served as controls. All rats that developed palpable P-SV tumors were
autopsied, and surviving rats were autopsied at age 24 months.
RESULTS. At age 24 months, 9 of 70 DHT-treated rats (12.8%) and 20 of 75 DHT-free control
rats (26.6%) had developed P-SV tumors spontaneously at average age 20.5 and 20 months,
respectively.
CONCLUSIONS. Slow-release implants of DHT administered to L-W rats at age 12 months
reduced by 50% the development of spontaneous P-SV tumors by age 24 months. Prostate
36:168–171, 1998. © 1998 Wiley-Liss, Inc.
KEY WORDS:
dihydrotestosterone; prostate-seminal vesicle cancer; spontaneous tumors; prevention of P-SV tumors
INTRODUCTION
In 1987, we reported that testosterone propionate
(TP) promoted, but that dihydrotestosterone (DHT)
did not promote, the development of cancers in the
accessory sex glands [prostate and seminal vesicles
(P-SV)] of Lobund-Wistar (L-W) rats [1]. Compared
with TP-treated rats, the testes in DHT-treated rats
were significantly reduced in size, levels of testosterone in their serums were significantly reduced; and
within the time-frame of 14 months, there was no microscopic evidence of spermatogenesis. The ducts and
acini in the prostatic lobes were lined by single layers
of columnar and cuboidal cells. Among control rats,
24% of rats that were treated with multiple implants of
TP developed large metastasizing tumors. The DHTtreated rats showed neither gross nor microscopic evidence of a tumorigenic effect [1]. The inhibitory effect
© 1998 Wiley-Liss, Inc.
of DHT on induced P-SV tumors was confirmed in
Fischer [2] and in L-W rats [3].
L-W rats, at risk of developing P-SV tumors that
were induced by a combination of methylnitrosourea
(MNU) plus TP [4] were administered DHT or estradiol or were castrated, at a time midway in the estimated latency period [5]. Within a time-frame of 14
months, the tumorigenic process was significantly inhibited by DHT, by estradiol and by castration; however, the same treatments were of no benefit in rats
that were treated after palpable P-SV tumors had developed [5].
Contract grant sponsor: The Coleman Foundation; Contract grant
sponsor: The Scully Trust; Contract grant sponsor: University of
Notre Dame.
*Correspondence to: Morris Pollard, Lobund Laboratory, University
of Notre Dame, Notre Dame, IN 46556.
Received 10 November 1997; Accepted 23 March 1998
DHT Prevents Spontaneous P-SV Tumors
DHT, metabolized from TP by the action of 5areductase, was described as the trophic androgen in
initiating proliferative changes and tumors in the
prostate gland [6–8]. This finding was confirmed in
men whose tumor-free status was associated with deficiency in 5a-reductase [9], and in men who had been
castrated when young [10]. The inhibitory effects of
DHT reported by us [1,5], contrasting with the reported trophic effect of DHT (6–8), was clarified: DHT
(5a-Androstan-17b-ol-3-one) inhibited the development of induced P-SV tumors, but DHT propionate
promoted tumor development [3]. The trophic effect
was also demonstrated with DHT-benzoate (Sigma
Chemical Company, St. Louis, MO) [11].
Metastasizing P-SV adenocarcinomas developed
spontaneously in 26% of L-W rats, in an average latency period of 26 months [12]. Further examinations
of 220 aged L-W rats revealed that 57 rats (26%) developed large spontaneous P-SV tumors in average
latency of 26 months; and that smaller tumors could
be detected earlier by palpation at average age 20
months [11]. This long latency period and low frequency rate simulated the natural history of prostate
cancer in man. It was of interest to determine whether
DHT would inhibit the development of spontaneous
P-SV tumors from estimated midlife span. L-W rats at
12 months of age were treated with a single subcutaneous implant of DHT, and they were observed until
they reached 24 months of age. The results of tumor
development in DHT-treated and in untreated control
rats is described below.
MATERIALS AND METHODS
Animals
Pathogen-free L-W rats were propagated at random
in isolated air-conditioned rooms with 12/12 hour
light-dark cycles. They were maintained in plastic
cages on a bedding of wood shavings and fed a whole
grain diet (L-485, TekLad, Madison, WI). Although not
intentionally inbred through 56 generations, L-W rats
did not reject reciprocal skin transplants. The care of
our experimental animals was and is in accord with
the United States Public Health Service bulletin Guide
for The Care and Use of Laboratory Animals. This facility
is AAALAC-accredited.
Experimental Design
At age 12 months, 70 male L-W rats were implanted
subcutaneous with 40 mg of DHT (Sigma Chemical
Company), which was enclosed in a slow-release silicone membrane [1]. This implant of DHT exerted an
antigonadotropic effect for >14 months [1,5]. Seventy-
169
five control rats and 70 DHT-treated rats were fed ad
libitum whole grain diet L-485 (TekLad). They were
examined at frequent intervals for body weights, for
changes in sizes of palpable testes, and for P-SV tumors. Rats with palpable P-SV tumors were killed by
inhaled halothane and exsanguination from the heart;
then they were examined for gross and microscopic
lesions. All surviving rats were killed for similar examinations when they reached age 24 months. Tissue
specimens from DHT-treated and from control rats
were examined for histologic changes. The results
were assessed for significance by Student’s t- test and
by x2 test.
RESULTS
The DHT-treated and the control rats were in excellent physical condition, except that those with large
P-SV tumors lost weight. When rats were autopsied at
24 months of age, the implants of DHT were onequarter full.
Compared with data in control rats (Table I),
changes were noted in the DHT-treated rats recorded
here and are described in Ref. 1: (a) their palpable
testes were significantly reduced in size within 1
month, and, in rats that developed P-SV tumors, the
testes remained small for the duration of the experiment. (b) Serum testosterone levels were below the
level of detection (0.1 ng/ml ml serum) by the RIA test
used [5]. (c) At autopsy, the average weight of the
prostate complex in tumor-free rats was marginally
less than the weight in control rats. (d) The weight of
testes in DHT-treated rats was reduced by ∼70%, and
the seminiferous tubules were free of spermatozoa. (e)
The ducts and acini in the prostate and in the seminal
vesicles were clean and lined with single layers of columnar and cuboidal cells. The connective tissue stromata were relatively sparse. These changes were also
recorded and are described in Ref. 1. (f) There was
little structural evidence of the prostatic intraepithelial
neoplasia (PIN) described by Bostwick and Shrigley
[13]. (g) Palpable P-SV adenocarcinomas developed
spontaneously in 9 of 70 DHT-treated rats (12.8%) in
an average latency time of 20.5 months, and in 20 of 75
control rats (26.6%) in an average latency time of 20
months.
Also, pituitary glands and Leydig cells in the testes
were not altered in appearance and size in the DHTtreated rats, and there were no cytolytic changes in the
involuted testes. In a separate experiment, 1 month
after removal of the DHT implant from six rats, their
small testes were restored to original size, and their
breeding potency, previously negative, was restored
[11].
170
Pollard
TABLE I. Comparison of DHT-treated and Untreated Control
L-W Rats†
DHT treated
547
0.0*
3.06 ± 0.59*
0.31*
Negative*
Sparse
9 of 70 (12.8%)*
20.5 months
Results
Controls
Average body weight/g
Serum testosterone in ng/ml
Average prostate complex/g
Average testes weight/g
Spermatogenesis
PIN in prostate complex
P-SV adenocarcinomas
Average latent period/months
521
1.4
3.89 ± 0.82
1.38
Positive
Positive
20 of 75 (26.6%)
20 months
†
Male L-W rats, age 12 months, were administered one subcutaneous
implant of 40 mg of DHT enclosed in a slow-release silicone membrane.
DHT-treated and control untreated rats were autopsied on appearance of
tumors, and survivors were autopsied at age 24 months. The information
listed was derived from tumor-free DHT-treated and age-related control
untreated rats.
*Statistically significant.
DISCUSSION
There is growing consensus that gene-regulated
mechanisms govern predisposition to prostate cancer
[14]; but the actual development of tumors can be suppressed or promoted by factors in the environment.
Suppression of tumorigenesis in susceptible animals
would be manifested by absence of tumors, by reduced incidence of tumors, and/or by prolongation of
the latency time. A recent review on prostate cancer
recommended that three transplantable cell lines derived from metastatic tumors in men and one cell line
(Dunning) from a rat serve as models for studies on
pathogenesis of prostate cancer [15]. Pathogenesis is
defined as ‘‘the organization and development of a
disease.’’ We can agree that transplantable malignant
and metastatic tumor cells had basically completed the
sequence of pathogenic events leading to malignancy;
thus, malignant tumors are not models for studies on
pathogenesis. Decades of research on transplanted tumors have contributed little to our knowledge of the
tumorigenic process; and the research rarely produced
information that was confirmed through trials in animals with autochthonous tumors. This finding was
exemplified in the following trials in the L-W rat: (a)
cyclophosphamide (CPA) suppressed transplanted
PA-III cells, but CPA was inactive against the development of induced P-SV tumors [16]; (b) 4-hydroxyphenyl retinamide very significantly suppressed
metastatic spread of transplanted PA-III cells, but the
effects on induced autochthonous P-SV tumors was
marginal [17,18]. Transplantation models are being replaced by more authentic models of autochthonous
tumors, which are needed to assess data that would be
prerequisite to clinical trials.
L-W rats are genetically susceptible to the development of spontaneous and induced metastasizing adenocarcinomas in the prostate-seminal vesicle (P-SV)
complex. The development of induced P-SV tumors
was suppressed by early treatments with antiandrogenic agents and procedures [5], and with antigonadotropic DHT. The incidence of induced P-SV tumors
was reduced in L-W rats by antiangiogenic linomide
[3] and by tamoxifen [19]. Metastatic spread of autochthonous P-SV tumor cells was suppressed by viable
BCG organisms, only if BCG had been inoculated intravenously [20]. The latency periods, but not the incidence of induced tumors, was prolonged by feeding
experiments with a soybean-derived high isoflavone
diet [21]. The incidence of spontaneous tumors was
suppressed by life-long moderate dietary restriction
[22]; and in the experiment reported here on DHT the
incidence of spontaneous tumors was reduced by 50%
when treatment was initiated at 12 months of age. The
evidence is accumulating that the natural history of
the disease in L-W rats resembles facets of the epidemiologic pattern of prostate cancer in man [23] and
that the predicted altered ‘‘hormonal environment’’
may provide for early intervention in prostate cancer
[24]. The practicality of controlling prostate cancer in
man by an implant of DHT may not attract volunteers,
but DHT did prevent the hormone-influenced disease
in rats without overt persistent damage to the host.
There are unanswered questions in this report on
DHT: (a) what is the optimal dosage of DHT; (b) will
the incidence of tumors be further reduced by earlier
treatment of rats with DHT; (c) where in the hypothalamic-pituitary-testicular axis is the functional linkage
interrupted by DHT; (d) are the results on induced
DHT Prevents Spontaneous P-SV Tumors
P-SV tumors comparable to results on spontaneous
tumors; and (e) why was esterified DHT active but
nonesterified DHT inactive in the development of PSV tumors [3]? Acknowledging the limits of confidence in model systems, evidence in accumulating
that environmental (epigenetic) factors can modify
gene-directed prostate-related cancers.
ACKNOWLEDGMENTS
I acknowledge, with thanks, the important contributions to this work by Phyllis H. Luckert, and by
Valerie Schroeder of the Freimann Life Science Center.
10.
11.
12.
13.
14.
15.
16.
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