[CANCER RESEARCH 49, 5889-5894. November I. 1989]
Gene Activity during the Early Phase of Androgen-stimulated Rat Prostate
Regrowth1
Aaron E. Katz, Mitchell C. Benson, Gilbert J. Wise, Carl A. Olsson, Mark G. Bandyk, Ihor S. Sawczuk,
Philip Tomashefsky, and Ralph Buttyan
Department of Urology, Maimonides Medical Center, Brooklyn, New York 11219 [A. E. K., G. J. W.], and Department of Urology, Columbia
New York, New York 10032 [M. C. B., C. A. O., M. G. B., l. S. S., P. T., R. B.]
ABSTRACT
Androgenic steroids regulate the proliferation rate of normal and
malignant prostate cells. In order to investigate the molecular basis of
this control, we utilized Northern and Western blot techniques to measure
changes in the expression of individual genes during the early phase of
prostate regrowth. Vestigial ventral prostate glands of 7-day castrated
rats showed increased numbers of replicating cells within 12 h of contin
uous pharmacological testosterone replacement as demonstrated by flow
cytometric procedures. The period prior to the onset of proliferative
enhancement was accompanied by the sequential induction of a variety
of transcripts encoding gene products often associated with cell growth.
Within l h of treatment, mature mRNA transcripts for c-fos were induced
6-fold, returning to control levels by 2 h. Other genes showed transiently
elevated transcript levels after 2 h (c-Ha-ro?, c-Ki-ras) or after 8 h
(c-myc, c-myb, 0-actin, and M, 70,000 heat shock protein) of testosterone
replacement. Expression of the polypeptide encoded by c-I la-ra.v was
coordinately enhanced (2-fold) during this period, but not to the levels of
the transcript (20-fold induction). Transcripts encoding basic fibroblast
growth factor were increased 16 h and later, subsequent to the earlier
enhancement in the proliferation rate. By placing these genes in a defined
temporal order with regard to their expression in response to testosterone,
we have constructed a map of gene activity during early prostate regrowth.
This map shows a number of genes, the products of which might partic
ipate in the mechanism by which androgens induce mitogenesis of pros
tate cells.
INTRODUCTION
Androgenic steroids are characterized by their ability to reg
ulate the gene activity of androgen-sensitive cells. Like other
steroid hormones, androgens mediate their effect through an
intracellular receptor protein which may stimulate (or repress)
the expression of specific gene products, depending on the
nature of the target cell. For some cells, androgens also have a
mitogenic influence. As exemplified by the developing mam
malian prostate gland, testosterone will induce the rapid growth
of this organ by increasing the rate of cellular proliferation.
Currently, the relationship between the androgen stimulus and
the mitogenic response is unclear. Studies on other systems of
inducible cell growth have demonstrated that cell proliferation
is a complex process requiring the coordinated participation of
multiple gene products (1, 2). Thus, in the prostate gland, the
mitogenic effect of androgenic steroids is likely to be mediated
by the action of a battery of gene products expressed, either
directly or indirectly, under the influence of androgens.
This investigation was designed to increase our understand
ing of the molecular mechanism by which androgens regulate
prostate growth. We have begun to catalogue the acute changes
in gene activity that accompany androgen-stimulated prostate
cell proliferation in a regrowing rat ventral prostate model.
Using probes for gene products known to participate in cellular
Received 4/3/89; revised 7/21/89; accepted 8/4/89.
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.
1This work was partially supported by a grant from the Maimonides Research
and Development Foundation and from the NIH (CA47848) (R. B.).
University,
growth, we were able to quantitate the expression of a large
number of genes as a function of time following androgen
stimulation. The genes that we assayed for included a variety
of protooncogenes (c-fos, c-myc, Ha-ras, Ki-ras, and c-myb),
genes required for cellular maintenance and homeostasis (actin,
tubulin, protein kinase C, and M, 70,000 heat shock protein),
and genes encoding specific growth factors (c-sis and basic
fibroblast growth factor). By placing these genes in a defined
temporal order in terms of their acute response to testosterone,
we have been able to construct a preliminary map of gene
activity during androgen-stimulated proliferation of prostate
cells. Although the expression of many different gene products
increased during acute prostate regrowth, the intense response
of a few particular genes implies the possibility that their
products may drive the mitogenic response to androgens.
MATERIALS
AND METHODS
Androgenic Manipulation of Rats. Mature (350-375 g) male SpragueDawley rats (Camm Industries, Wayne, NJ) were surgically castrated
under sodium pentobarbitol anesthesia (35 mg/kg i.p.). Castrated rats
were maintained under standard laboratory conditions for 7 days to
allow regression of the ventral prostate gland. At this time, testosterone
was returned by an injection of testosterone propionate (2 mg/kg i.m.
in sesame oil). At various times following testosterone treatment,
groups of rats were sacrificed with sodium pentobarbitol (100 mg/kg).
Ventral prostate tissue was removed and frozen in liquid nitrogen for
later RNA and protein extraction. Rats maintained for longer than 12
h were given repeated injections at 12-h intervals to maintain pharma
cological testosterone levels during the length of the experiment (up to
60 h).
Flow Cytometric Analysis of Cell Cycle Components during Testoster
one Stimulation of Prostate Regrowth. Ventral prostate glands were
collected from groups of rats at 7 days after castration and at specified
hourly intervals after testosterone stimulation of 7-day castrated rats.
Individual glands were mechanically minced into 1-mm3 pieces and the
cells were dispersed with gentle stirring in a trypsin-EDTA solution
(Sigma Chemical Co., St. Louis, MO). Following digestion, samples
were passed through cotton gauze and were centrifuged at 3000 rpm
for 10 min. The cell pellet was washed with Dulbecco's modified Eagle's
medium containing 10% fetal calf serum and then incubated in a
propidium iodide solution (50 ^g/ml) containing 4 HIMsodium citrate
(pH 7.8), 30 units/ml DNase-free RNase, and 0.1% Triton X-100.
Flow cytometric measurements were performed using a Coulter EPICS
752 flow cytometer (argon laser, 488 nm) equipped with a dedicated
M DADS computer. For each analysis 50,000 nuclei were counted.
PARA 1 analysis of cell cycle components was used. By this means,
the percentage of nuclei in the G0 + d, S, and G2 + M phase of each
sample was determined. The coefficient of variation for every sample
analyzed remained below 5.0.
Extraction of Poly(A)+2 mRNA and Quantitation of Transcript Levels.
Pooled groups of frozen ventral prostate glands from 5 rats/time point
were pulverized in liquid nitrogen and the powder was homogenized in
a solution of 5 M guanidine ¡sothiocyanate-5% 2-mercaptoethanol. As
described previously (3), RNA was precipitated from the homogenate
2The abbreviations used are: poly(A)* mRNA, polyadenylated mRNA; SDS,
sodium dodecyl sulfate; cDNA, complementary DNA; bFGF, basic fibroblast
growth factor; p21, M, 21.000 protein.
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GENE ACTIVITY DURING PROSTATE REGROWTH
with LiCl and purified by phenolxhloroform extraction. Poly(A)+
mRNA was isolated by oligodeoxythymidylate cellulose chromatography (Boehringer Mannheim Biochemicals, Indianapolis, IN). RNA
concentration was quantitated by spectrophotometry at 260 uni. Sam
ples (10 jig) of poly(A)+ RNA were electrophoresed in 1.2% agaroseformaldehyde gels at 50 V for 18-20 h and were transferred to nitro
cellulose filter by capillary blotting overnight. RNA quality was assessed
by the migration and intensity levels of ethidium-bromide stained
residual rRNA bands combined with the sharp quality of the transcript
bands detected following hybridization. Blots were prehybridized in a
solution of 6 x standard saline citrate (1 x standard saline citrate is
0.15 M NaCI-0.015 M sodium citrate), 5 mM EDTA, 0.5% polyvinylpyrrolidone, 0.5% bovine serum albumin, 0.5% Ficoll, 0.5% SDS, 50%
deionized formamide, and 100 /¿g/mldenatured salmon sperm DNA
for 4 h. Hybridization continued with the addition of a denatured 32Plabeled probe, as described, for 18 h at 42°C.Probes were radiolabeled
using nick translation (4) in the presence of [a-32P]deoxyribonucleotides. Following hybridization, blots were washed in a successive series
of solutions containing 0.1% SDS, 1 mM EDTA, and decreasing
amounts of standard saline citrate (from 2 x to 0.1 x) at 55°C.Blots
were exposed to Kodak XAR-5 X-ray film with fluorographic screens
at —¿70"C.
Quantitative densitometry was performed on autoradiograms
using a computer-based measurement of the integral of area and density
for each band on a Joyce-Loebel Chromoscan 3 densitometer.
Probes for Northern Blot Analysis. The probes utilized for Northern
blot analysis were obtained from the following sources: /3-actin and atubulin cDNA (5) from Donald Cleveland (Johns Hopkins University,
Baltimore, MD); \-fos (6) from Thomas Curran (Roche Institute of
Molecular Biology, Nutley, NJ); murine c-myc (third exon) (7) from
Frederick Alt (Columbia University, New York, NY); v-Ha-ras, Ki-roj,
c-myb, and c-sis from Oncor, Inc. (Gaithersburg, MD); c-kinase (RP58) (8) from Bernard Weinstein (Columbia University, New York, NY),
heat shock 68 cDNA (9) from Larry Moran (University of Toronto,
Toronto, Ontario, Canada); 18S rRNA cDNA from Ramreddy Guntaka
(University of Missouri, Columbia, MO); cDNA for bFGF obtained
from Judy Abraham (California Biotechnology, Inc., Mountain View,
CA); and cDNA for C3 (10) from Malcolm Parker (Imperial Cancer
Research Laboratories, London, England).
Analysis of I Ia-rt/v Protein Levels during Prostate Regrowth. Ventral
prostate tissue from 7-day castrated rats and regrowing ventral prostates
recovered at 4, 8, 12, 16, and 20 h after testosterone replenishment
were minced in phosphate-buffered saline and homogenized in a buffer
containing 50 mM Tris-HCl (pH 7.5), 10 mM NaCl, 1 mM MgCl2,
0.5% Triton X-100, and 5% sucrose with a Dounce homogenizer.
Following centrifugation at 5000 x g, the supernatant fraction was
recovered and stored at -20°C. Western blot analysis of the extracts
established the quality of the unti Ha-n/.v p21 antiserum used for
immunoquantitation. Samples containing 100 /¿g
protein were electro
phoresed on denaturing 12% SDS-polyacrylamide gels according to the
method Laemmli (11). Proteins in the gel were transferred to a nitro
cellulose filter by electroelution at 125 mA for 18 h in 10% methanol25 mM Tris-HCl, pH 7.5-0.2 M glycine. The Western blot was blocked
in 10% powdered milk for l h and then incubated with sheep anti-Ka
ra v p21 antiserum (Triton Biosciences, Alameda, CA) at a 1:100 dilu
tion. Antibody binding was detected using secondary rabbit anti-sheep
IgG labeled with alkaline phosphatase (Kirkegaard and Perry Labs,
Inc., Gaithersburg, MD). Alkaline phosphatase activity was visualized
with 5-bromo-4-chloro-3-indolyl phosphate-p-toluidine and nitroblue
tetrazolium chloride substrates at pH 9.5. Relative quantitation of Ila
ras p21 levels in extracts was accomplished through slot-blot immunodetection. Aliquots of prostate cytosol containing 2.5, 5, and 10 Mg
of protein were applied to a nitrocellulose filter with a slot-blot appa
ratus. The filter was treated the same as for the Western blot. Quanti
tation of alkaline phosphatase staining in individual slots was per
formed by a video-densitometer apparatus using the ImageMeasure
program (Microscience, Inc., Federal Way, WA).
RESULTS
Changes in the Flow Cytometric Parameters of the Regrowing
Rat Ventral Prostate Consistent with the Early Onset of DNA
Synthesis. We chose to study the regrowth of the regressed rat
ventral prostate gland as a model system for androgen-stimulated cell proliferation. Castration of a mature rat is accom
panied by the regression of the ventral prostate gland. By
returning androgens, however, the vestigial remnant of the
ventral prostate is stimulated to regrow back to the normal
adult size. In order to establish the time at which DNA synthesis
can be induced in ventral prostate tissue in response to testos
terone, we monitored the flow cytometric pattern of ventral
prostate nuclei as a function of the time following testosterone
treatment of long-term castrated rats. Groups of 7-day castrated
rats received testosterone (2 mg every 12 h) and at various
times following treatment, individual ventral prostate glands
from groups of 4-5 rats were processed and analyzed for cell
cycle components by quantitive flow cytometry. Fig. 1 indicates
the mean percentage of replicating cells (S -I- G2M) in the
glands from each group of rats. After 8 h of testosterone
treatment, the number of replicating cells in individual glands
increased significantly (P< 0.005) so that at 60 h posttreatment,
approximately 34% of the prostate cells are replicating. It is of
interest that the groups in the transition period (8 and 12 h
after treatment) show large standard errors compared to the
pretreatment, early treatment, and late treatment groups, per
haps reflecting individual variation in the time at which the rats
in these groups begin to show a response to the treatment.
Changes in Transcript Levels Reveal Gene Activity during the
Acute Period of Prostate Regrowth. Regrowing ventral prostate
glands harvested at designated time intervals after testosterone
treatment of 7-day castrated rats were processed to extract
poly(A)* mRNA. These RNAs were analyzed by Northern blot
methods to quantitate the expression of transcripts homologous
to a variety of gene probes, as indicated. Individual blots were
reprobed at least 3 times to allow the analysis of the expression
of a large number of genes during the first 30 h after testoster34
32
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HOURS AFTER
12
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20
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TESTOSTERONE
56
60
REPLACEMENT
Fig. 1. Flow cytometric quantitation of replicating cells in androgen-stimulated rat ventral prostate glands. Individual rat ventral prostate glands removed
at various time intervals following testosterone replacement of 7-day castrated
rats were enzymatically dispersed into single cell suspensions and were incubated
in a hypotonie butler containing propidum iodide. Flow cytometric evaluation of
cell cycle components was performed and the distribution of nuclei (as a percent
age of nuclei counted) into the G0 + G,, S and G2M phase was determined by
PARA-1 computer analysis. The percentage nuclei in S + G2M was used as an
index of replicating cells. Points, mean number of replicating cells in each time
group; bars, SE calculated for each group.
5890
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GENE ACTIVITY DURING PROSTATE REGROWTH
one stimulation. Typical hybridization patterns are shown by
the photographs of Northern blot autoradiograms for 6 of these
probes (Figs. 2 and 3). Relative transcript levels for each gene
probed were determined by quantitative densitometry of indi
vidual transcript bands at each time point. These numbers were
corrected for potential deviation in RNA loading levels by
comparing them to the time-equivalent densities of 18S ribosomal bands obtained when the blot was hybridized to a probe
for 18S rRNA. The change in relative transcript band densities
for 6 different genes are shown Fig. 4. The rapid induction of
mRNA for C3 confirms the effectiveness of testosterone treat
ment (Figs. 3 and 4). C3 encodes a subunit of the major
secretory protein of the rat ventral prostate and its expression
is highly dependent on the levels of circulating testosterone
(10).
By this method, we were able to group the genes analyzed
into 4 different categories: rapid response genes (within 2-3 h
of testosterone replacement), as shown by c-fos, c-Ha-ras, c-Kiras, and C3; intermediate response genes (within 6-8 h of
testosterone replacement), as exemplified by c-myc, c-myb,
actin, hsp-70; late response genes (16 h after testosterone
replacement), as demonstrated by bFGF; and nonresponsive
genes (as shown by protein kinase C and c-sis). Utilizing this
hierarchy of gene activity in response to testosterone, we have
constructed a temporal gene map (Fig. 5) demonstrating the
time course of the molecular changes expressed by prostate
cells during androgen-stimulated
proliferation. These early
changes in transcript levels are consistent with a previous study
which demonstrated a rapid increase in RNA polymerase activ
ity by regrowing rat prostate (12). Other aspects of this acute
gene activity are the transient nature in the expression of many
HOURS AFTER T-REPLACEMENT
30
-2.5
Kb
hsp-68
-0.6 Kb
8
12
24
-1.8Kb
/B —¿actin
Fig. 2. Representative Northern blot analysis for gene expression during
regrowth of the rat ventral prostate gland. Poly(A)* mRNA was extracted from
groups of rat ventral prostate glands collected at various hourly intervals, as
indicated, following testosterone (T) replacement of 7-day castrated rats. The
RNA was electrophoresed and transferred to a nitrocellulose filter. As shown
above, the Northern blot was repeatedly hybridized to a series of radiolabeled
probes including probes for .-¡actin.C3, and M, 68,000 heat shock protein (hsp68). Kb, kilobases.
HOURS AFTER
O
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2
3
T-REPLACEMENT
4
8
16 24 30
—¿
I.I Kb
cHa-ras
—¿
1.9Kb
bFGF
—¿
2.0Kb
I8S rRNA
Fig. 3. Representative Northern blot analysis of genes expressed during regrowth of the rat ventral prostate gland. Poly(A)* mRNA extracted from groups
of rat ventral prostate glands collected at various time intervals, as indicated,
following testosterone (T) replacement of 7-day castrated rats. Northern blots
made from this RNA were probed repeatedly for the expression of a variety of
genes including c-Ha-ras, bFGF or for the levels of residual 18S rRNA to control
for loading equivalency. Kb, kilobases.
of the genes, c-fos is the most notable in this regard. In a
manner similar to that of other systems of induced proliferation
(13, 14), c-fos is the first gene that shows response to testoster
one stimulus in the rat ventral prostate. It is also the most brief
response, demonstrating a 6-fold induction within the first hour
after testosterone replacement, but returning to control levels
by the second hr. In addition to the different times at which the
various genes show changes in response to testosterone, is the
different intensity of response by each gene. Of all of the genes
we analyzed, transcripts encoding c-Ha-ras show the most
striking levels of enhancement in response to testosterone stim
ulation, increasing as much as 20-fold within the first 4 h after
testosterone replacement. Although not to the apparent degree
of the c-Ha-ras transcript, transcripts encoding c-Ki-ros also
shows a high level of induction (Fig. 4).
Increased Synthesis of Protein p21 c-Ha-ras during Prostate
Regrowth. mRNA transcripts are the primary gene products in
the pathway to gene expression. In order to determine whether
the changes in the level of transcripts that we observed reflect
the increased synthesis of any of the corresponding proteins,
we analyzed for the expression of one particular gene product,
p21 c-Ha-ras, in cytosol extracts of regrowing ventral prostates.
We utilized a commercially available antiserum against this
protein to immunoquantitate the levels of p21 c-Ha-ras as a
function of the time after testosterone replacement. Western
blot analysis of a cytosol extract of a mature rat ventral prostate
showed that this antiserum recognized a M, 21,000 polypeptide
corresponding to the normal c-Ha-ras gene product (Fig. 6).
Equal amounts of protein from ventral prostate extracts at
various times after testosterone repletion were loaded into the
wells of a slot blot and were absorbed onto a nitrocellulose
filter. The filter was probed with anti-p21 c-Ha-ras and then
5891
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GENE ACTIVITY DURING PROSTATE REGROWTH
46
44
C3
40
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HOURS AFTER TESTOSTERONE REPLACEMENT
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8 10 12 14 16 IB 20 22 24 26
HOURS AFTER TESTOSTERONE REPLACEMENT
HOURS AFTER TESTOSTERONE REPLACEMENT
24
Fig. 4. Densitometric quantitation of changes in relative transcript levels over time in RNA of regrowing rat ventral prostate glands. Quantitative densitometry
was performed on autoradiographs of Northern blots probed for the expression of specific genes, as indicated. Values obtained from each band were corrected for
potential deviations in RNA loading by comparison to the relative density of the 18S rRNA band at that same time. Shown are graphical presentations of 6
representative genes.
PROSTATE
REGROWTH
A B
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TESTOSTERONE
REPLACEMENTl
SYNTHESIS24
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OR NO CHANGE:
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HYPERTROPHY
20
Fig. 5. Temporal map of gene activity during androgen-stimulated regrowth
of the rat ventral prostate gland. A variety of genes were ordered in terms of the
time of their induced expression in response to androgen stimulation of a
regressed rat ventral prostate. Arrows, time at which transcript levels for specified
genes begin to show changes following testosterone replacement of 7-day castrated
rats. Also shown is the period during which proliferation increases as a result of
androgen stimulation and the onset of hypertrophie changes in epithelial cells of
the regrowing ventral prostate gland as determined by our microscopic evaluation
of thin sections of fixed regrowing ventral prostate glands.
I
I -2lkd
10
0
4
8
HOURS AFTER
c-Sis
C-kmase
'
12
16
20
T-REPLACEMENT
Fig. 6. Expression of p21 c-Ha-ras protein in regrowing rat ventral prostate
glands. As shown by comparison to molecular weight standards (A) on a Western
blot, antiserum against p2l c-Ha-ras recognized a M, 21,000 (_'/ kd) polypeptide
in a cytoplasmic extract of the rat ventral prostate gland (B). Left, Slot-Western
blot analysis of cytosol extracts from regrowing ventral prostate glands. Each slot
contained S *ig protein extracted from ventral prostate glands at hourly intervals
after testosterone (T) replacement of 7-day castrated rats as indicated to the left.
Video-densitometric quantitation of this slot-Western blot allowed the relative
estimation of p21 c-Ha-raj protein levels in regrowing prostates (center).
DISCUSSION
with alkaline phosphatase-labeled secondary antibody to detect
binding of the primary antibody. Colorimetrie substrate detec
tion gives a relative estimate of the amount of primary antibody
bound in each slot and thus reflects the relative amounts of p21
in the samples. By this method, we estimate that the amount
of p21 c-Ha-ras protein per mg of protein in regrowing prostate
cytosol extracts increased at least 2-fold over the 20-h period
after testosterone repletion (Fig. 6). While this change does not
reflect the high level induction of the transcript, it does show
that increased expression of the ras protein product accompa
nies the induction of c-Ha-ras mRNA transcripts during pros
tate regrowth. If the production of proteins from the other
induced transcripts is similarly muted, however, the increased
expression of their protein products may be difficult to detect
by these kinds of batch techniques.
Androgenic steroids can exert a potent mitogenic effect on
prostate cells in vivo. To date, the molecular mechanism of this
stimulation remains poorly defined. Several aspects of the
prostate gland make analysis of the androgen-potentiated pro
liferation response difficult. First, the mitogenic effect of androgens vary according to the development of the organ. Tes
tosterone stimulation can dramatically accelerate the proliferative rate of prostate cells in a sexually immature rat, yet once
the organ has attained adult size, additional testosterone has
little influence (15). As our model system for analysis, we
utilized the regrowing rat ventral prostate gland. In a manner
similar to that for the immature prostate gland, a fully regressed
(vestigial) ventral prostate in a mature, castrated rat responds
to androgen stimulation with the rapid onset of cell replication
(IS). In prior studies, rad iolabeled thymidine incorporation had
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GENE ACTIVITY DURING PROSTATE REGROWTH
been used to monitor and time DNA synthesis in regrowing rat
prostate glands (16-19). These studies had shown large in
creases in DNA synthesis at a period 40 h or later following
testosterone stimulation. Our use of flow cytometric techniques
to analyze prostate regrowth has allowed us to detect a much
earlier increase in the number of replicating cells starting after
testosterone replacement for 8 h.
The utility of thymidine incorporation measurements as a
means to show changes in the proliferation rate is dependent
on equivalent uptake of radiolabeled thymidine by the different
cells of the organ and also by the size of the endogenous pools
of thymidine in various cellular compartments. Flow cytometric
quantitation of cell cycle components is not influenced by either
of these factors, suggesting that we may be measuring an early
response to androgens by ventral prostate cells resistant to the
uptake or incorporation of exogenous thymidine. The early
period of proliferation in a regrowing prostate would be con
sistent with our ability to detect a concomitant rapid increase
in the expression of genes associated with cell growth and
would suggest that the products of these genes participate in
the mechanism by which androgens stimulate prostate cell
proliferation.
The prostate gland is composed of a complex mixture of
cells, each of which responds in different degrees to androgens.
Thus the question of whether androgens act directly on each
cellular component of the organ, or whether, by stimulating
one individual cell type, androgens indirectly stimulate the
proliferation of the other cells in the gland remains to be
determined. Relevant to this question is the recent burst of
research on prostatic growth factors. Studies of cultured cells
and growing tissues have shown that cell proliferation is often
regulated by the availability of polypeptide growth factors (19).
Thus various investigators have suggested that androgens might
control prostate growth in either a paracrine or an autocrine
manner by inducing the expression of a prostate-specific growth
factor (20-22). At least 2 different growth factors have been
detected in rat prostate tissue and have been shown to have
similarity to bFGF and epidermal growth factor (23, 24). While
we have not yet probed regrowing rat prostate tissue for the
expression of epidermal growth factor-like molecules, our stud
ies of bFGF indicate that this growth factor is synthesized by
the rat prostate gland and that the expression of this gene is
induced by testosterone stimulation. However, the apparent
induction of transcripts for bFGF occurs at a time after we see
changes in the number of replicating cells in the gland (after 12
h), suggesting that if bFGF does play a role in prostate regrowth
it is secondary to growth initiated by other effects of testoster
one replacement. Considering the angiogenic properties of
bFGF (25), it is possible that the delayed expression of this
growth factor is related to a demand for an increased blood
supply to the growing regions of the prostate.
Finally, the complex nature of the stromal and epithelial cell
element interaction must be considered when evaluating the
significance of any gene expression study in the prostate. Al
though the stromal cells consist of only 10-15% of the cellular
population of the ventral prostate gland, reconstitution experi
ments suggest that these cells are the major regulators of
glandular growth potential in response to androgens (26). None
of the experiments which we have described here helps us
discern whether the early onset of proliferation and gene activity
is restricted to either the stromal or epithelial cells. As shown
by prior in situ studies in which radiolabeled thymidine was
used to selectively label replicating cells, the later phase of
DNA synthesis (after 45 h) involves mainly glandular epithelial
and basal cells ( 18). A similar approach or other in situ methods
of gene expression analysis might help us distinguish whether
the early period of DNA synthesis is restricted to a particular
cellular compartment.
Cell proliferation is a complex action, regardless of the
stimulus. The genes that we report on in this study represent
only a small fraction of the potential pool of known genes that
might be involved in androgen stimulation of prostate cell
growth. However, we believe that in this restricted study, we
have identified several candidate gene products that might
mediate prostate cell proliferation responses to androgens. The
early and brief induction of the c-fos gene product is a landmark
seen in other systems of inducible proliferation (13, 14). c-fos
activity is also an early marker of the onset of cellular differ
entiation and even programmed cell death (3, 27). While the
induction of this particular gene product during all these various
modes of cellular response might argue against its specific
participation in any given response, cells that express antisense
c-fos mRNA are blocked in their ability to proliferate (28, 29).
Therefore the early induced expression of c-fos products might
be required for the prostate cell to respond to and process
extracellular signals (androgens) in an appropriate manner.
As a secondary event, we have detected the rapid and intense
induction of transcripts encoding genes of the ras family cor
responding with a smaller but significant increase in the expres
sion of the p21 ras protein. Abberant expression of raj genes
has been linked to abnormal growth of tissues and cells during
oncogenesis (30, 31), and the intense stimulation in the activity
of these genes in prostate cells by androgens might clearly play
a role in driving these cells to synthesize DNA. Alternatively,
however, we must consider that the expression of the c-Ha-ras
gene has also been shown to be intensely activated during
cellular hypertrophy as demonstrated during compensatory regrowth of a rodent renal model (32). Since one of the early
responses of the ventral prostate gland to testosterone replace
ment is a hypertrophy of the surviving epithelial cells, perhaps
the induced expression of the ras genes also help mediate this
response. Finally, three of the genes we analyzed (c-myc, ßactin, and a-tubulin) showed increased expression closer to the
onset of DNA synthesis. This finding is of interest since the
products of these genes are more likely to have a direct role in
cell division. The product of the c-myc gene is believed to play
a role in DNA synthesis (33) while the actins and tubulins are
important for the formation of the mitotic apparatus and cy
tokinesis.
The complexity of the response of the prostate gland to
androgens is reflected in the large number of genes the expres
sion of which fluctuates during androgenic stimulation. While
the map of gene activity during androgen-stimulated prolifera
tion is far from complete, it does define several genes of interest.
Identifying the specific gene products involved in prostate pro
liferation may allow us to devise new strategies to impede
abnormal prostate growth.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the expert technical assistance
of Po-Ying Ng and Frieda Karp.
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Gene Activity during the Early Phase of Androgen-stimulated
Rat Prostate Regrowth
Aaron E. Katz, Mitchell C. Benson, Gilbert J. Wise, et al.
Cancer Res 1989;49:5889-5894.
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