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 Laboratories, a Division of Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN; J. Baniel, Department of Urology, Indiana University School of Medicine, Indianapolis; P. Cohen, Division of Pediatric Endocrinology, Children'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, Indianapolis, IN 46285.
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ABSTRACT |
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INTRODUCTION |
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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, preventing 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, 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 activity (13-15) produced almost exclusively by prostatic epithelia. Because of its tissue specificity and association with prostatic proliferative disorders, PSA has become a useful serum marker in the management of prostatic cancer. Numerous clinical 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 development of the genitourinary tract and the pathogenesis of BPH (19-21). Evidence obtained from prenatal animal studies supports an inductive role for the fibromuscular stroma in epithelial proliferation and phenotypic expression. Few studies have investigated the inductive capabilities of epithelial cells on fibromuscular 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.
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MATERIALS AND METHODS |
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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 temperature.
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-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 x 4.6 mm; Applied Biosystems, Foster City, CA), equilibrated in 0.1%
trifluoroacetate 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 purified PSA was examined with the use of a high-performance liquid chromatography (HPLC) (Beckman System Gold, Fullerton, CA) assay. Briefly, 357 nM purified PSA was incubated with 72 µM of a 10-amino-acid synthetic peptide substrate (SGAWYYVPLG) in the presence of various concentrations (0-10 µM) 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 x 250 mm, 5 µm; The Separations Group, Hesperia, CA) with a gradient mobile phase consisting of 0.1% trifluoroacetate with increasing concentrations 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 confirmed by review of histologic sections of representative tissue specimens. Stromal 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 µg/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 1216 hours. The dissociated stromal and epithelial cells were separated with the use of discontinuous PercollTM (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 µg/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 identified 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 plates (Falcon, Bedford, MA) at a concentration of 1 x 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 = 2637 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 µg/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 µg/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 evaluate effects on cellular responses to PSA. BPH-derived stromal cells were cultured as described above for 48 hours in the presence of 100 µg/mL PSA and various concentrations of ZnCl2 (0, 0.1, 1.0, 10, and 100 µM). 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 experiments. 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.
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RESULTS |
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IGF-I stimulated BPH-derived stromal cells in a
concentration-dependent manner (Fig. 1). After 48
hours of treatment, a medium concentration of 10 ng/mL IGF-I produced a
47% increase in the numbers of BPH-derived stromal cells above control
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 containing bovine serum
albumin for periods longer than 48 hours resulted in cell death.
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Exogenously added IGFBP3 inhibited IGF-I-induced stimulation of
BPH-derived stromal cells in a concentration-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 numbers
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.
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At concentrations up to 100 µg/mL, PSA stimulated modest
proliferation of BPH-derived stromal cells (data not shown). In these
same experiments after a 48-hour treatment, 100 µg/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).
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Fig. 4 illustrates the mitogenic effects of IGF-I
(10 ng/mL) and PSA (100 µg/mL) on BPH-derived stromal cells (P =
.00001 and P = .0036, respectively). Also shown is the
inhibition of the growth of IGF-I-stimulated BPH-derived stromal cells
by IGFBP3 (100 ng/mL; P = .0002). Addition of PSA (100
µg/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 1618 hours abolished the indirect proliferative effects
of PSA (P = .00001). Preincubation of ZnCl2 with
IGF-I had no 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).
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-Chymotrypsin was cytotoxic to BPH-derived stromal cells at
concentrations equal to or greater than 10 µg/mL (data not shown).
Overall,
-chymotrypsin at a concentration of 3 µg/mL had a
slight stimulatory (8.7% ± 1.2% greater than control) effect on
the growth of BPH-derived stromal cells, whereas concentrations lower
than 3 µg/mL had no effect. Addition of
-chymotrypsin to
cultures of BPH-derived stromal cells containing IGF-I and IGFBP3
restored the stimulatory effect of IGF-I on BPH-derived stromal cells
(data not shown).
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DISCUSSION |
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Epidemiologic observations document that BPH and prostatic cancer are major factors affecting the health of the male population in the United States (30). Despite the importance of these diseases, our current perceptions of the cellular aberrations responsible for the development of BPH and prostatic cancer remain 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 multifaceted 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 cancer and responses to therapy have been well documented. However, PSA serum levels may also be elevated in BPH (16). In addition, several investigators (37-40) have shown that the concentrations of intraprostatic PSA are significantly higher in patients with BPH than in patients with prostatic cancer. These published observations provide evidence that the prostatic fibromuscular 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 circulation 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 activity of PSA on these carrier proteins. Our findings are consistent with
the possibility that the prostatic stroma may be exposed 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 patients 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 regulatory PSA enzyme activity. Reductions in tissue zinc concentrations have been reported in prostatic cancer patients (46,47). In addition, the concentration of zinc is significantly lower in stromal 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 enzymatically active PSA to enhance cellular proliferation. These findings provide further clinically relevant associations supporting regulation of enzymatically active PSA, which may be involved 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 different 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 surrounding 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 laboratory. 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 prostate epithelial cells to PSA.
From the above evidence, we conclude that PSA may play both a direct and an indirect role in stimulating prostatic fibromuscular stromal cells. These observations are consistent with the concept that PSA may play a physiologic role in the regulation of prostatic cell growth. However, many issues remain to be addressed before a definitive conclusion regarding this possibility can be reached. Additional experimentation will be necessary to determine the role of PSA in stimulating normal and neoplastic accessory sex organ growth in vivo.
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NOTES |
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Manuscript received May 26, 1999; revised July 30, 1999; accepted August 6, 1999.
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