Growth Regulation of Prostatic Stromal Cells by
Prostate-Specific Antigen
Debra M. Sutkowski, Robin L. Goode, Jack Baniel, Carroll Teater, Pinchas Cohen,
Ann M. McNulty, Hansen M. Hsiung, Gerald W. Becker, Blake Lee Neubauer
Background: Prostate-specific antigen (PSA) is a serine pro-
tease that can cleave insulin-like growth factor-binding pro-
tein-3 (IGFBP3), thereby decreasing its affinity for insulin-
like growth factor-I (IGF-I). Dissociation of the IGF-I–
IGFBP3 complex renders IGF-I available to bind to its
receptor and stimulates cellular proliferation. We evaluated
the potential for PSA to modulate the effects of IGF-I and
IGFBP3 on the proliferation of human benign prostatic hy-
perplasia (BPH)-derived fibromuscular stromal cells in pri-
mary cultures. Methods: We cultured BPH-derived stromal
cells for 48 hours in serum-free RPMI-1640 medium supple-
mented with 0.2% bovine serum albumin and studied the
effects of IGF-I, IGFBP3, PSA, and ZnCl2 at varying con-
centrations. Differences in cell growth between control and
treated cultures were evaluated by use of Dunnett’s test.
Concentration-related trends were evaluated by linear re-
gression of log-transformed concentrations of test reagents
on BPH-derived stromal cell number responses. Statistical
tests were two-sided. Results: We observed a concentration-
dependent proliferative response of BPH-derived stromal
cells to IGF-I. IGFBP3 inhibited this response in a concen-
tration-dependent fashion. IGFBP3 alone had no effect on
stromal cell proliferation. When stromal cells were incu-
bated with PSA alone or with PSA, IGF-I, and IGFBP3, an
increase in stromal cell numbers that was dependent on PSA
concentration was evident in both instances. Zinc, an endog-
enous inhibitor of PSA enzymatic activity, was able to at-
tenuate the stimulatory effect of PSA at intraprostatic physi-
ologic concentrations. Conclusions: These results are
consistent with the idea that PSA can modulate in vitro in-
teractions between IGF-I and IGFBP3 and suggest that PSA
may play a role in the regulation of human prostatic fibro-
muscular cell growth. [J Natl Cancer Inst 1999;91:1663–9]
Peptide growth factors are potent regulators of cellular pro-
liferation and metabolic functions. Insulin-like growth factor-I
(IGF-I) is synthesized in many tissues and stimulates the growth
of both benign and malignant cells (1). IGF-I has been postu-
lated to act as an autocrine or paracrine growth factor for a
variety of malignant cells, including prostatic cancer cells (2–4).
The mitogenic effects of IGF-I are modulated in a positive and
negative manner by IGF-binding proteins (IGFBPs) (5–7). At
least seven species of IGFBPs have been identified and charac-
terized (8,9).
The exact mechanism(s) by which IGFBPs regulate the action
of IGF molecules are not fully elucidated. However, evidence
supports the idea that the IGFBPs can interact with IGF, pre-
venting the growth factor from binding to its plasma membrane
receptor. Proteases are thought to regulate the biologic activity
of IGFBPs (10,11).
It has been demonstrated that prostate-specific antigen (PSA)
is capable of cleaving IGFBP3, lowering its affinity for IGF-I,
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which allows IGF-I to bind to its membrane receptor on benign
prostatic hyperplasia (BPH) epithelial cells (11,12). PSA is a
serine protease that possesses chymotrypsin-like enzymatic ac-
tivity (13–15) produced almost exclusively by prostatic epithe-
lia. Because of its tissue specificity and association with pros-
tatic proliferative disorders, PSA has become a useful serum
marker in the management of prostatic cancer. Numerous clini-
cal investigations (16,17) have sought to define the importance
of PSA in the early detection of prostatic cancer. In seminal
plasma, PSA has been identified as the enzyme responsible for
proteolysis of semenogelin, resulting in the liquefaction of the
seminal gel. Seminal liquefaction is a process obligatory to the
release of progressively motile spermatozoa (18). While seminal
liquefaction activity has been defined for PSA, its physiologic
role(s) in prostatic fluid remains undefined. The fibromuscular
stromal component of the prostate and of other accessory sex
organs is thought to play an inductive role in the fetal develop-
ment of the genitourinary tract and the pathogenesis of BPH
(19–21). Evidence obtained from prenatal animal studies sup-
ports an inductive role for the fibromuscular stroma in epithelial
proliferation and phenotypic expression. Few studies have in-
vestigated the inductive capabilities of epithelial cells on fibro-
muscular proliferation.
The experiments described in this article were carried out to
determine a possible paracrine role of PSA in the growth of
human prostatic fibromuscular stromal cells.
MATERIALS AND METHODS
Reagents
Recombinant human IGF-I for biologic experiments was produced at Lilly
Research Laboratories (Indianapolis, IN). Recombinant human IGFBP3 was
purchased from UBI (Lake Placid, NY) and resuspended in 10 mM acetic acid.
␣-Chymotrypsin was purchased from Sigma Chemical Co. (St. Louis, MO).
Enzymatically active PSA was purified from human seminal plasma with the use
of a three-column chromatography technique. Human semen was incubated at
37 °C for 20 minutes followed by centrifugation of the liquefied material in a
Sorvall RC5C centrifuge (Kendro Laboratory Products, Newtown, CT) with the
use of an SS34 rotor at 10 000 rpm (12 000g) for 15 minutes at room tempera-
ture. The resulting seminal plasma was loaded onto a Superose 6 (10/30) column
(Pharmacia Biotech Inc., Piscataway, NJ) equilibrated in 100 mM ammonium
bicarbonate (pH 7.8) and run at a flow rate of 0.5 mL/minute. The PSA-
containing fractions were identified with the use of sodium dodecyl sulfate–
Affiliations of authors: D. M. Sutkowski, R. L. Goode, C. Teater, A. M.
McNulty, H. M. Hsiung, G. W. Becker, B. L. Neubauer, Lilly Research Labo-
ratories, a Division of Eli Lilly and Company, Lilly Corporate Center, India-
napolis, IN; J. Baniel, Department of Urology, Indiana University School of
Medicine, Indianapolis; P. Cohen, Division of Pediatric Endocrinology, Chil-
dren’s Hospital of Philadelphia, PA.
Correspondence to: Blake Lee Neubauer, Ph.D., Cancer Research 0546, Lilly
Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, India-
napolis, IN 46285.
See “Notes” following “References.”
© Oxford University Press
ARTICLES 1663
 polyacrylamide gel electrophoresis (SDS–PAGE). The PSA-positive fractions
were combined, adjusted to pH 5.5 with the use of 0.1 N HCl, and loaded onto
a Mono S (5/5) column (Pharmacia Biotech Inc.) equilibrated in 50 mM malonic
acid (pH 5.5). After a brief period of isocratic flow at initial conditions, protein
was eluted from the column with a linear gradient of NaCl in 50 mM malonic
acid (pH 5.5) (0–500 mM NaCl; 1.0 mL/minute; approximately 30 minutes).
PSA-containing fractions were again localized with the use of SDS–PAGE,
combined, and loaded onto a reversed-phase column (Aquapore RP-300; 250 ×
4.6 mm; Applied Biosystems, Foster City, CA), equilibrated in 0.1% trifluoro-
acetate and 10% acetonitrile. Protein was eluted at a flow rate of 1.0 mL/minute,
with the use of a linear gradient of acetonitrile increasing at 1% per minute. All
columns in this purification scheme were monitored spectrophotometrically at
214 nm. The purified protein was exchanged into phosphate-buffered saline
(PBS) (pH 7.4) with the use of a Sephadex G-25 column. N-terminal sequence
analysis was performed with a model 470A or a model 477A protein sequencer
(Applied Biosystems), and the N-terminal sequence was determined to be IVGG.
The molecular mass of the purified protein was found to be 28 430 by analysis
on a triple quadrapole mass spectrometer fitted with an electrospray ionization
source (API-III; PE-Sciex, Toronto, ON, Canada).
PSA–Zinc Interaction
The ability of zinc chloride (ZnCl2) to inhibit the enzymatic activity of puri-
fied PSA was examined with the use of a high-performance liquid chromatog-
raphy (HPLC) (Beckman System Gold, Fullerton, CA) assay. Briefly, 357 nM
purified PSA was incubated with 72 ␮M of a 10-amino-acid synthetic peptide
substrate (SGAWYYVPLG) in the presence of various concentrations (0–10
␮M) of ZnCl2 for 2 hours at 37 °C. PSA cleaved this substrate between the two
tyrosine residues. To terminate the reaction, we added trifluoroacetate to the
mixture to yield a final concentration of 0.1%. The peptide fragments were
separated by HPLC with the use of a Vydac C-18 column (protein and peptide
column, 4.6 × 250 mm, 5 ␮m; The Separations Group, Hesperia, CA) with a
gradient mobile phase consisting of 0.1% trifluoroacetate with increasing con-
centrations of acetonitrile (15%–30%) over a 30-minute period. Results were
expressed as the area under the peak representing absorbency of substrate and
peptide fragments detected at 280 nm.
Cell Cultures
Human prostatic stromal cells were isolated from tissues obtained from three
patients undergoing transurethral prostatectomy procedures for treatment of
bladder neck obstruction secondary to BPH. The diagnosis of BPH was con-
firmed by review of histologic sections of representative tissue specimens. Stro-
mal cells used in these studies have been characterized and used in previously
published studies (22,23). Surgical specimens of the prostate were pre-
dissociated enzymatically in RPMI-1640 medium containing 10% fetal bovine
serum (Life Technologies, Inc. [GIBCO BRL], Gaithersburg, MD), 100 ␮g/mL
deoxyribonuclease (Sigma Chemical Co.), and 200 U/mL type I collagenase
(Sigma Chemical Co.) for a period of 4 hours. The supernatant containing red
blood cells and debris was discarded. The prostatic specimens were rinsed three
times with PBS, mechanically dissociated into pieces of approximately 1 mm3 in
size with the use of surgical scalpels, and further dissociated enzymatically for
a period of 12–16 hours. The dissociated stromal and epithelial cells were sepa-
rated with the use of discontinuous Percoll™ (Sigma Chemical Co.) gradient
centrifugation for 30 minutes at 500g (25 °C). Stromal cells were selectively
cultured in RPMI-1640 medium without phenol red, supplemented with 10%
fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 ␮g/mL)
(Sigma Chemical Co). Cells were cultivated in T75 tissue culture flasks under
routine conditions. Immunocytochemical analysis of the purified stromal cells
used in these studies revealed the preparations to be composed of both smooth
muscle cells and fibroblasts. The predominant smooth muscle cells were iden-
tified by use of an antibody directed against alpha-smooth muscle actin (antibody
A2547 [1 : 500 dilution]; Sigma Chemical Co.) and fibroblasts stained positively
for prolyl 4-hydroxylase (antibody 631631 [1 : 500 dilution]; ICN Biomedicals,
Inc., Costa Mesa, CA). Typical cultures contained up to 99% of cells staining
positively for alpha-smooth muscle actin (24,25).
In Vitro Growth Assays
BPH-derived stromal cells obtained from passages 4–12 were dissociated
from culture flask surfaces by brief treatment with trypsin–EDTA (0.25%) (Life
Technologies, Inc.) and washed. Cells were reseeded in 24-well tissue culture
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plates (Falcon, Bedford, MA) at a concentration of 1 × 104 cells per well in
phenol red-deficient RPMI-1640 medium containing 10% fetal bovine serum
and penicillin and streptomycin as described above. The mean doubling time for
the BPH-derived stromal cells obtained from the three specimens was 32 hours
(range ⳱ 26–37 hours). Cells were allowed to adhere overnight. The stromal
cells were washed the following day with PBS, and experimental reagents were
applied in basal medium that consisted of RPMI-1640 (without phenol red)
supplemented with 2 mg/mL bovine serum albumin (Sigma Chemical Co.) and
a 1% (vol/vol) penicillin (104 U)–streptomycin (104 ␮g/mL) solution. After 48
hours, cells were harvested and cell numbers were determined with the use of a
model 901 cell counter (Coulter Corp., Miami, FL).
Experimental Conditions
The concentration-dependent mitogenic activity of IGF-I (0, 1, 3, 10, 30, and
100 ng/mL) was assessed by the measurement of the in vitro proliferation of
BPH-derived stromal cells with the use of the culture conditions described
above. The optimal stimulatory concentration of IGF-I was determined, and the
ability of IGFBP3 to modulate the mitogenic effect of IGF-I on the growth of
BPH-derived stromal cells was examined. IGFBP3 (0, 30, 100, 300, and 500
ng/mL) and IGF-I (10 ng/mL) were added simultaneously to cells growing in
culture, and cell numbers were determined as described previously.
After defining the maximal inhibitory concentration of IGFBP3 on IGF-I-
induced stromal cell proliferation, we evaluated the ability of purified PSA (0, 3,
10, 30, 100, and 300 ␮g/mL) to modulate this inhibition. Similar concentrations
of PSA were examined for independent effects on the cells and served as controls
for the IGF-I and IGFBP3 combination experiments.
Zinc has been shown to be an endogenous inhibitor of the enzymatic activity
of PSA (26). ZnCl2 was added to stromal cell cultures containing PSA to evalu-
ate effects on cellular responses to PSA. BPH-derived stromal cells were cul-
tured as described above for 48 hours in the presence of 100 ␮g/mL PSA and
various concentrations of ZnCl2 (0, 0.1, 1.0, 10, and 100 ␮M). PSA and ZnCl2
were preincubated for 24 hours at 4 °C to allow association of these two reagents
before these agents were applied to the cells in culture. Similar concentrations of
ZnCl2 were examined for independent effects on the stromal cells. Levels of
IGF-I, PSA, IGFBP3, and ZnCl2 determined in the above concentration–
response studies to produce marked stimulatory or antagonistic actions were
tested in combination to evaluate their interaction on the proliferation of BPH-
derived stromal cells.
Statistical Analysis
Unless otherwise stated, results for in vitro experiments are expressed as
means ± standard error of four observations. The data shown are representative
results of consistent qualitative responses observed in triplicate or more experi-
ments. Data were analyzed for significant differences at the P<.05 level with the
use of Dunnett’s test (27) and for normal distribution by the Shapiro–Wilk W test
(28). Concentration-related trends were evaluated by linear regression of log-
transformed concentrations of test reagents on BPH-derived stromal cell number
responses. Statistical tests were two-sided.
RESULTS
IGF-I Stimulation of Proliferation of BPH-Derived
Stromal Cells
IGF-I stimulated BPH-derived stromal cells in a concentra-
tion-dependent manner (Fig. 1). After 48 hours of treatment, a
medium concentration of 10 ng/mL IGF-I produced a 47% in-
crease in the numbers of BPH-derived stromal cells above con-
trol levels (P ⳱ .0015). Maximal cell numbers were observed
with IGF-I concentrations equal to or greater than 30 ng/mL.
Culture of BPH-derived stromal cells in defined medium con-
taining bovine serum albumin for periods longer than 48 hours
resulted in cell death.
IGFBP3 Antagonism of Proliferation of BPH-Derived
Stromal Cells Induced by IGF-I
Exogenously added IGFBP3 inhibited IGF-I-induced stimu-
lation of BPH-derived stromal cells in a concentration-
 Fig. 1. Insulin-like growth factor-I (IGF-I) stimulates the growth of primary
cultures of benign prostatic hyperplasia (BPH)-derived stromal cells in serum-
free medium in a concentration-dependent fashion. The stromal cells (1 × l04/
well) were plated in 24-well plates. After adherence overnight (16–18 hours), the
medium was changed to RPMI-1640 containing 0.2% bovine serum albumin and
varying concentrations of IGF-I. The cells were detached from the culture sur-
face after 48 hours with 0.25% trypsin and counted with the use of a Coulter
counter. Significantly greater than no IGF-I control (*P ⳱ .0015; **P ⳱
.00004; ***P ⳱ .00001; ****P ⳱ .00001) with the use of Dunnett’s test (27).
Values represent the means ± standard error of the mean of four observations
from one of three representative experiments.

dependent fashion (Fig. 2, A). Simultaneous exposure of BPH-

derived stromal cells to IGF-I (10 ng/mL) and IGFBP3 (100
ng/mL) resulted in statistically significant decreases in numbers
of BPH-derived stromal cells compared with cultures treated
with IGF-I (P ⳱ .0005). The cell numbers (mean ± standard
error of the mean) from IGF-I cultures inhibited by co-
incubation with IGFBP3 (300 ng/mL) were not significantly
different from the values for control cells in the presence of
bovine serum albumin (P ⳱ .102). The higher concentration of
IGFBP3 did not produce further inhibition than that produced by
100 ng/mL IGFBP3. IGFBP3-induced reductions in cell num-
bers were not attributable to the binding protein per se (Fig. 2,
B). Despite a slight trend toward increased stromal cell numbers
(P ⳱ .079), IGFBP3 had no statistically significant effect on the
growth of BPH-derived stromal cell cultures after a 48-hour
incubation.
Stimulation of Proliferation of BPH-Derived Stromal Cells
by PSA
At concentrations up to 100 ␮g/mL, PSA stimulated modest
proliferation of BPH-derived stromal cells (data not shown). In
these same experiments after a 48-hour treatment, 100 ␮g/mL
PSA stimulated statistically significant increases in numbers of
BPH-derived stromal cells above control values (P ⳱ .0311)
(Fig. 3, A). ZnCl2, an endogenous inhibitor of PSA enzymatic
activity, was able to abolish the stimulatory activity of PSA at all
concentrations tested (Fig. 3, A).
While a slight trend toward increased stromal cell numbers (P
= .088) was observed, ZnCI2 had no effect on the growth of
BPH-derived stromal cells up to a concentration of 10 ␮M (Fig.
3, B). Table 1 describes inhibition of PSA enzymatic activity by
ZnCl2 in a concentration-dependent manner with the use of a
biochemical assay. The amount of substrate (SGAWYYVPLG)
cleaved by PSA after 2 hours of incubation was lower in the
Fig. 2. A) Insulin-like growth factor-binding protein-3 (IGFBP3) inhibits insu-
lin-like growth factor-I (IGF-I)-induced stimulation of primary cultures of be-
nign prostatic hyperplasia (BPH)-derived stromal cells in a concentration-
dependent fashion. Cells were incubated under routine culture conditions for 48
hours in the presence of 10 ng/mL IGF-I and various concentrations of IGFBP3
(0–300 ng/mL). * ⳱ significantly greater than control (no additions) (P ⳱
.00001) and significantly less (**P ⳱ .0005; ***P ⳱ .00002; ****P ⳱ .00001)
than cultures treated with only IGF-I with the use of Dunnett’s test (27). B) The
same concentrations of IGFBP3 in basal medium in the absence of IGF-I had no
effect on proliferation of BPH-derived stromal cells. Values represent the means
± standard error of the mean of four observations from one of three represen-
tative experiments.
presence of ZnCl2. Similar concentrations of CaCl2 (10.0 ␮M)
had no effect on the ability of PSA to cleave the substrate.
PSA and Inhibition by IGFBP3 of Cellular Proliferation
Induced by IGF-I
Fig. 4 illustrates the mitogenic effects of IGF-I (10 ng/mL)
and PSA (100 ␮g/mL) on BPH-derived stromal cells (P ⳱
.00001 and P ⳱ .0036, respectively). Also shown is the inhibi-
tion of the growth of IGF-I-stimulated BPH-derived stromal
cells by IGFBP3 (100 ng/mL; P ⳱ .0002). Addition of PSA
(100 ␮g/mL) to cultures containing IGF-l and IGFBP3 restored
the stimulatory effects of IGF-l on the proliferation of BPH-
derived stromal cells (P<.00001). Preincubation of ZnCl2 with
PSA for 16–18 hours abolished the indirect proliferative effects
of PSA (P ⳱ .00001). Preincubation of ZnCl2 with IGF-I had no

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Fig. 3. A) Prostate-specific antigen (PSA) (100 ␮g/mL) stimulates the growth of
benign prostatic hyperplasia (BPH)-derived stromal cells in culture. Intrapros-
tatic physiologic concentrations of zinc attenuate the mitogenic effect of PSA on
primary cultures of BPH-derived stromal cells. PSA (l00 ␮g/mL) and ZnCl2
were preincubated for 24 hours at 4 °C to allow mixing of these two reagents and
applied to BPH-derived stromal cells in culture for 48 hours to evaluate the effect
of zinc on PSA-induced cellular proliferation. B) Similar concentrations of
ZnCl2 had no independent effects on cellular proliferation up to 10 ␮M (P for
trend ⳱ .088). * ⳱ significantly greater than control (no additions) (P ⳱ .0311)
and significantly less than cultures treated only with PSA (**P ⳱ .0005; ***P
⳱ .0014; ****P ⳱ .0023) with the use of Dunnett’s test (27). Values represent
the means ± standard error of the mean of four observations from one of three
representative experiments.
effect on IGF-I-induced cellular proliferation (data not shown).
In addition, ZnCl2 had no effect on IGFBP3 inhibition of IGF-
I-induced stimulation of BPH-derived stromal cells (data not
shown).
␣-Chymotrypsin Concentration–Response Study
␣-Chymotrypsin was cytotoxic to BPH-derived stromal cells
at concentrations equal to or greater than 10 ␮g/mL (data not
shown). Overall, ␣-chymotrypsin at a concentration of 3 ␮g/mL
had a slight stimulatory (8.7% ± 1.2% greater than control)
effect on the growth of BPH-derived stromal cells, whereas con-
centrations lower than 3 ␮g/mL had no effect. Addition of
␣-chymotrypsin to cultures of BPH-derived stromal cells con-
taining IGF-I and IGFBP3 restored the stimulatory effect of
IGF-I on BPH-derived stromal cells (data not shown).
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Table 1. Effect of ZnCl2 on ability of prostate-specific antigen (PSA) to
cleave a 10-amino-acid substrate between two tyrosine residues*
% inhibition of
Treatment group Peak area† PSA enzymatic activity†
Control 92 812 ± 1387 0
0.1 ␮M ZnCl2 90 498 ± 1715 4.25 ± 1.1
1.0 ␮M ZnCl2 73 660 ± 806 20.4 ± 1.2
10 ␮M ZnCl2 67 889 ± 861 27.2 ± 1.4
10 ␮M CaCl2 92 212 ± 1804 0
*PSA was incubated with substrate (SGAWYYVPLG) for 2 hours at 37 °C in
the presence of various concentrations of ZnCl2. CaCl2 served as a nonspecific
control with no effect on PSA enzymatic activity.
†Values (absorbance units) represent the means ± standard error of the mean
of five observations from one of two representative experiments.
Fig. 4. Prostate-specific antigen (PSA) antagonizes insulin-like growth factor-
binding protein-3 (IGFBP3)-induced inhibition of insulin-like growth factor-I
(IGF-I) stimulation. IGF-I (10 ng/mL) stimulated growth of benign prostatic
hyperplasia (BPH)-derived stromal cells as indicated by the increase in cell
number after 48 hours. Simultaneous addition of IGF-I (10 ng/mL) and IGFBP3
(100 ng/mL) did not produce a substantial increase in cell number. Addition of
PSA (100 ␮g/mL) to the culture medium attenuated the effect of IGFBP3,
restoring the mitogenic properties of IGF-I. ZnCl2 (1 ␮M) abolished the indirect
proliferative effect of PSA. *P ⳱ .0036 and **P ⳱ .00001 were significantly
greater than control (no additions); *** significantly (P<.009) less than IGF-I-
stimulated cells; ****significantly (P<.00001) greater than IGF-I + IGFBP3-
treated cells; *****significantly (P<.00001) less than IGF-I + IGFBP3 + PSA-
treated cells, with the use of Dunnett’s test (27). Values represent the means ±
standard error of the mean of four observations from one of three representative
experiments.
DISCUSSION
Results of our study show that IGF-I stimulated in a concen-
tration-dependent fashion the proliferation of the BPH-derived
stromal cells obtained from three separate specimens grown in
tissue culture. IGFBP3 inhibited IGF-I-stimulated growth of
BPH-derived stromal cells, and the inhibitory effect of IGFBP3
was attenuated by the proteases PSA and ␣-chymotrypsin. PSA
alone was also able to directly stimulate the growth of BPH-
derived stromal cells in vitro by an average of 17% above con-
trol cell numbers within a 48-hour incubation. ␣-Chymotrypsin
was only able to stimulate proliferation of BPH-derived stromal
cells on an average of approximately 9% above control cell
numbers within the same time period. This finding is consistent
 with the hypothesis that the serine protease activity associated
with both enzymes may only be a part of the stimulatory func-
tion of PSA in the proliferation of BPH-derived stromal cells.
While the magnitude of PSA-induced cellular proliferation ob-
served in our assays is relatively modest when compared with
the IGF-I responses, the protease stimulated concentration-
dependent increases in cell numbers. Prostatic stromal cells
make insulin-like growth factors and IGFBP3 (29). Exogenous
PSA may be proteolyzing the stromal cell-derived IGF-I–
IGFBP3 complex, producing apparent direct stimulatory re-
sponses. Whatever the mechanism of PSA-induced stromal pro-
liferation, the magnitude of the response is consistent with the
relatively low rates of cellular proliferation in benign and ma-
lignant prostatic neoplasia. In addition, human prostatic stromal
cells will not be exposed to ␣-chymotrypsin, since this protease
is localized to the gut. ␣-Chymotrypsin is also cytotoxic to pros-
tatic stromal cells at concentrations much lower than the con-
centration of PSA used to stimulate proliferation of BPH-
derived stromal cells. These observations support the idea that
PSA may play a role in selectively regulating prostatic fibro-
muscular proliferation.
Epidemiologic observations document that BPH and prostatic
cancer are major factors affecting the health of the male popu-
lation in the United States (30). Despite the importance of these
diseases, our current perceptions of the cellular aberrations re-
sponsible for the development of BPH and prostatic cancer re-
main primitive. BPH results from the proliferation of prostatic
acinar, ductal, and stromal elements (31). Several investigators
(20,21) have suggested that the cellular inductive interactions by
stromal cells on epithelial tissue elements play a dynamic role in
modulating the normal and neoplastic growth of the prostate.
The stromal–epithelial inductive interactions are likely multifac-
eted events, which involve cell–cell contact, extracellular matrix
elements, and the production of soluble mediators (32–34). Our
data provide evidence for a potential stimulatory induction of
stromal cell proliferation by the paracrine actions of PSA, an
epithelial, androgen-dependent cellular secretory protein.
PSA is a serine protease belonging to the family of glandular
kallikreins (15). PSA is a glycoprotein regulated by androgens
and produced almost exclusively by prostatic epithelium
(13,35,36). The observations by Cohen et al. (12) that IGFBP3
is an in vivo substrate for PSA provide evidence supporting a
paracrine role for PSA on prostatic stromal cells. The clinical
utilities of PSA for monitoring the progression of prostatic can-
cer and responses to therapy have been well documented. How-
ever, PSA serum levels may also be elevated in BPH (16). In
addition, several investigators (37–40) have shown that the con-
centrations of intraprostatic PSA are significantly higher in pa-
tients with BPH than in patients with prostatic cancer. These
published observations provide evidence that the prostatic fibro-
muscular stroma may proliferate when exposed to intraprostatic
concentrations of PSA.
PSA enzymatic activity is regulated by several factors. In the
serum of healthy males, 85%–95% of the circulating PSA is
bound to either ␣1-antichymotrypsin or ␣2-macroglobulin (41).
Changes in the ratio of bound PSA to free PSA occur in the
serum of patients with BPH and prostatic cancer. PSA is bound
to ␣1-antichymotrypsin at a higher proportion in the circula-
tion of untreated prostatic cancer patients relative to BPH
patients. The association of PSA in the circulation with ␣1-
antichymotrypsin and ␣2-macroglobulin requires the enzymatic
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activity of PSA on these carrier proteins. Our findings are con-
sistent with the possibility that the prostatic stroma may be ex-
posed to increased levels of biologically active PSA before it
enters the circulation. Evidence also exists for an association
between PSA levels and levels of IGFBPs in the serum of pa-
tients with prostatic cancer (42). Elevated levels of serum PSA
are associated with high levels of serum IGFBP2 and reduced
levels of serum IGFBP3 compared with levels in healthy male
control subjects. This observation supports the observation by
Cohen et al. that IGFBP3 is an in vivo substrate for PSA. In
addition, it has recently been shown that increased levels of
IGF-I are associated with an increased risk of prostatic cancer
(43).
Free zinc has been identified as the factor responsible for the
antibacterial activity of normal prostatic fluid (44). Zinc has
been shown to serve as an in vivo defense mechanism against
prostatic invasion and subsequent urinary tract infections in men
(45). In addition to the antibacterial role of zinc in prostatic
tissues, we have provided evidence for a role of zinc in regula-
tory PSA enzyme activity. Reductions in tissue zinc concentra-
tions have been reported in prostatic cancer patients (46,47). In
addition, the concentration of zinc is significantly lower in stro-
mal than in epithelial preparations from BPH specimens (48).
These lower concentrations of zinc in prostatic cancer patients
and stromal tissue may increase the local availability of enzy-
matically active PSA to enhance cellular proliferation. These
findings provide further clinically relevant associations support-
ing regulation of enzymatically active PSA, which may be in-
volved in prostatic cellular proliferation. Before entering the
circulation, PSA is exposed to the surrounding prostatic stroma
in BPH and cancer tissues. This transient exposure may induce
prostatic fibromuscular stromal proliferation by at least two dif-
ferent mechanisms. The first mechanism for PSA induction of
cellular growth can be derived from its action in seminal fluid as
described previously by Cohen et al. (12). Intraprostatic PSA
may alter the affinity of IGFBP3 for IGF-I found in the sur-
rounding growth milieu, releasing the IGF-I and allowing it to
bind to its plasma membrane receptor on fibromuscular cells to
stimulate cellular proliferation. An alternative proliferative
mechanism might result from PSA binding to a specific cell
surface receptor. The proteases, thrombin, and nerve growth
factor have been demonstrated to exert some of their biologic
activity through specific cell surface receptor interactions (49–
51). This hypothesis is currently under investigation in our labo-
ratory. PSA may also work as an autocrine growth factor on the
surrounding responsive prostatic epithelia. Findings of Cohen et
al. (52) and our laboratory (53) demonstrate both direct and
indirect in vitro growth responses of normal and neoplastic pros-
tate epithelial cells to PSA.
From the above evidence, we conclude that PSA may play
both a direct and an indirect role in stimulating prostatic fibro-
muscular stromal cells. These observations are consistent with
the concept that PSA may play a physiologic role in the regu-
lation of prostatic cell growth. However, many issues remain to
be addressed before a definitive conclusion regarding this pos-
sibility can be reached. Additional experimentation will be nec-
essary to determine the role of PSA in stimulating normal and
neoplastic accessory sex organ growth in vivo.
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NOTES
At the time that these studies were conducted, D. M. Sutkowski, R. L. Goode,
C. Teater, A. M. McNulty, H. M. Hsiung, G. W. Becker, and B. L. Neubauer
were employees of Eli Lilly and Company. They own that company’s stock as
part of their compensation and retirement plan participation. P. Cohen is a
member of the speaker’s bureau for Eli Lilly and Company.
Manuscript received May 26, 1998; revised July 30, 1999; accepted August 6,
1999.
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