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* Institute for Biomedical Aging Research, Austrian Academy of Sciences, Innsbruck;
Ludwig-Boltzmann Institute for Andrology and Urology, Vienna; and
Department of Urology, University of Vienna, Austria
1Correspondence: Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, A-6020 Innsbruck, Austria. E-mail: peter.berger{at}oeaw.ac.at
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: SMP • aging • HPLC analysis • cAMP • mitochondria • Par-4 • p53
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Furthermore,
clinical prostatic cancer (PCa) develops in 1 in every 11 white males
and 1 in every 10 black males in the U.S. during their lifetimes,
representing the most common cancer in the male after lung cancer
(2)
.
Although the etiology of prostatic proliferative disorders is unknown,
development and function of the prostate are thought to be mainly under
endocrine control. Epithelial development is stimulated and secretory
function is maintained by continuing presence of serum testosterone,
which must be converted by the prostatic 5-
reductase into
dihydrotestosterone. Recently, it has been shown that pituitary-derived
prolactin (hPRL) and growth hormone influence prostatic growth and
function (3
4
5
6)
. In addition, auto/paracrine factors
deriving from stromal and/or epithelial cells are important regulators
of prostatic growth (7)
. Fibroblast growth factors,
epidermal growth factor (EGF), transforming growth factors and ß
(TGF-, TGF-ß), and insulin-like growth factors and their
corresponding binding proteins (IGFs, IGFBPs) provide homeostasis of
cell replication and cell death, which seems to be disturbed in elderly
men.
Prostatic epithelial cells secrete numerous proteins and other substances into the seminal plasma (SMP). We postulate that such luminal factors, originally needed for optimal fertility, act on prostate epithelial cells by influencing growth, differentiation, and secretory function. Moreover, luminal factors beneficial for reproduction in young men could promote aberrant prostatic cell growth in the elderly.
SMP predominantly contains secretions from sex accessory tissue, with
the major contributions from seminal vesicle and prostate. In relation
to other body fluids, SMP is unique because of its very high
concentrations of zinc (150 µg/ml), citric acid (4 mg/ml), fructose
(2 mg/ml), spermine (3 mg/ml), prostaglandins (200 µg/ml), and
proteins (40 mg/ml) (8)
. SMP contains powerful
immunosuppressive agents considered to prolong the life of spermatozoa
and to prevent hypersensitization of the female to proteins present on
the surface of sperms (9)
. SMP from normal fertile men
revealed a pattern of over 200 proteins ranging in molecular mass from
10 to 100 kDa (10)
. Most proteins originate from the
prostate (8)
, like prostate-specific antigen prostatic
acid phosphatase, prostate-specific protein (PSP-94), and Zn- 2
glycoprotein. Furthermore, neuroendocrine peptides (somatostatin and
serotonin) and thyrotropin-releasing hormone-like peptides have been
described in SMP (11)
.
The aim of this study was to isolate and analyze factors from human SMP regulating prostate epithelial cell proliferation, viability, and differentiation. SMP was fractionated by size exclusion high-performance liquid chromatography (HPLC) and single fractions analyzed for growth stimulatory, inhibitory, and apoptotic effects. Furthermore, SMP-derived substances inducing apoptosis in prostate epithelial cells were compared in their actions and cell death pathways to the classic apoptotic stimulus, UV irradiation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1 h),
spermatozoa were removed by centrifugation (1000 g, 20 min,
4°C) and the supernatants were stored at -20°C for up to 8 wks.
Samples were diluted 1:2 with RPMI 1640 (BioWhittaker, Verviers,
Belgium) containing 10 mg/ml penicillin, 100 units/ml streptomycin, and
10 mg/ml L-glutamine (PSG) and pooled.
BPH-1 cells were obtained from the German Collection of Microorganisms
and Tissue Cultures (DSMZ, Braunschweig, Germany). This cell line has
been derived from the epithelium of prostatic tissue obtained from a
65-year-old Caucasian patient suffering from benign prostatic
hyperplasia. The primary culture was immortalized with the SV-40 large
T antigen, and one isolated clone was designated BPH-1
(12)
.
Both androgen-independent PC 3, derived from a primary prostatic tumor,
and androgen-dependent LnCAP, isolated from prostate cancer metastasis,
were obtained from the American Tissue Type Culture Collection
(Rockville, Md.). All cell lines were cultured in RPMI 1640 containing
5% bovine calf serum (BCS, A-2151-L, Hyclone, Logan, Utah) and PSG.
Human prostate epithelial cells were obtained from a patient (68 years)
suffering from hormonally untreated prostatic cancer. After radical
prostatectomy a cube of 0.125 cm3 was removed
from an area containing no histological signs of tumor. After
mechanical disruption, small organoids (1–5 mm3) were
cultured on Biocoat® collagen type I-coated discs (Becton Dickinson,
Vienna, Austria). Organoids were cultured in RPMI 1640 containing PSG
and 10% BCS for a least 7 days. Most organoids attached and were
surrounded by outgrown cells of epithelial origin, which stained
positive for the marker cytokeratin 8/18 (Autogen Bioclear, Wiltshire,
U.K.).
HPLC analysis of SMP
SMP was diluted 1:2 with RPMI 1640 containing PSG and
centrifuged at 12,000 g for 30 min (4°C). Supernatants
were filtered (Anotop R 10, 0.2 µm; Merck, Germany) to remove
residual sperm, loaded on a Sephadex column (Superdex 200®, Amersham
Pharmacia Biotech, Uppsala, Sweden), and eluted with phosphate-buffered
saline (PBS, 0.05 M phosphate, 0.15 M NaCl) at 0.4 ml/min. Fractions of
0.4 ml were collected and the eluate was monitored at 280 nm. A
molecular weight (MW) gel filtration calibration kit (Amersham
Pharmacia Biotech) containing blue dextran 2000 (2000,000 Da), mouse
IgG (160,000 Da), bovine serum albumin (67,000 Da), ovalbumin (43,000
Da), chymotrypsinogen A (25,000 Da), ribonuclease A (13,700 Da), and
prostaglandin E1 (360 Da) was used to estimate SMP protein sizes.
Proliferation/cell viability assays
Cells were seeded at a density of 5000 cells/well into a 96-well
microtiter plate and, after adherence (5 h), were incubated with SMP
(0.1–3.2%) in the presence of serum or under serum-free conditions.
The effects of prostaglandins (PGE-1, PGE-2), dihydrotestosterone, and
ß-estradiol (Sigma Chemical, St. Louis, Mo.) were analyzed at
concentrations ranging from 10-5 to
10-8.
Cell viability, i.e., activity of mitochondrial succinate dehydrogenase, was determined by cleavage of the tetrazolium salt WST-1 to formazan (Boehringer Mannheim, Mannheim, Germany) after 24 and 48 h. Each well was incubated with a 1:10 dilution of WST-1 for 30 min and the optical density (OD) was determined at 450 nm in a multiwell spectrophotometer (ELISA reader, Dynatech MR 5000).
DNA synthesis (proliferation) was determined by a slightly modified 5-Bromo-2'deoxy-uridine (BrdU) labeling and detection kit (Boehringer Mannheim). In brief, cells were incubated for 2 h with 10 µM BrdU, fixed, and permeabilized at -20°C for 20 min with a chilled buffer consisting of 70% ethanol and 50 mM glycine (pH 2). Thereafter, they were incubated with 0.5 µg/ml antibody directed against BrdU for 1 h. Detection was performed by the use of biotinylated anti-mouse immunoglobulin, streptavidin HRP conjugate (Dako, Denmark), and a peroxidase substrate set consisting of Sigma FastTM 3,3-diaminobenzidine tetrahydrochloride with metal enhancer tablets (Sigma).
Annexin V/propidium iodide (PI) staining and
fluorescence-activated cell sorting (FACS) analysis
Log-phase growing BPH-1 cells (1x106)
seeded into 6-well plates (40% confluence) were incubated with 2%
SMP under growth stimulation with 1% BCS. In parallel, control cells
were irradiated with UV light (1 kJ/m2). Cells
were harvested after 2, 4, 6, and 10 h by centrifugation (300
g, 5 min, 4°C), washed with ice-cold PBS, and resuspended
in 1 ml binding buffer (10 mM HEPES/NaOH pH 7.4, 1.4 mM NaCl, 2.5 mM
CaCl2). One hundred microliters of each cell
suspension was incubated with 5 µl annexin V-FITC (PharMingen, San
Diego, Calif.) and 10 µl PI (50 µg/ml) for 15 min in the dark. Then
cells were analyzed by flow cytometry (FACScan, Becton Dickinson) and
data were processed by the use of the Cell Quest software (Becton
Dickinson).
For cellular DNA fragmentation analyses, UV-irradiated (1 kJ/m2) and SMP-incubated (2%) cells were fixed and permeabilized with ice-cold 70% EtOH, washed in PBS, stained with propidium iodide solution (10 µg/ml, 10 min), and subsequently analyzed by flow cytometry.
Fluorescence staining of nucleus and cytoskeleton
Log-phase growing cells were cultivated in glass chamber slides
(Labtech® Nalco, Nunc International, Naperville, Ill.) and thereafter
exposed to 1% SMP. For staining of cytoskeleton and nuclei, cells were
fixed with 4% paraformaldehyde/PBS for 20 min and permeabilized for 10
min with 0.2% Triton X-100 in PBS. Chamber slides were rinsed with PBS
(10 min) and incubated with 100 ng/ml TRITC-labeled phalloidin
dissolved in PBS (visualizing filamentous actin, Sigma), 1 ng/ml Sytox
green (Molecular Probes, Eugene, Oreg.), or 100 ng/ml 4'
2-diamine-2'-phenylindole dihydrochloride (DAPI, Boehringer Mannheim),
respectively. Cells were washed twice with PBS for 5 min and either
viewed on a fluorescent microscope (Leitz) and photographed with Kodak
Ektachrome 160T or investigated by the µ-Radiance confocal scanning
(Bio-Rad, Hercules, Calif.). Mitochondria were stained with the JC-1
mitochondrial potential sensor (Molecular Probes). Log-phase growing
BPH-1 cells were cultivated in glass chamber slides and exposed to 1%
SMP for 2, 4, and 6 h. After extensive washing in culture medium,
cells were incubated with 1 µg/ml JC-1
(5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine-iodide,
Molecular Probes) for 30 min, washed twice (PBS), embedded in
culture medium, and immediately viewed on the confocal scanning system.
Western blots
Log-phase growing BPH-1 cells (4x106)
were seeded into a plate; after adherence, culture medium was changed
to RPMI 1640 containing 1% BCS. Cells were either incubated with 1%
SMP or UV irradiated (1 kJ/m2). Whole cell
extracts were prepared after 3, 6, and 9 h of incubation (37°C)
by lysing each sample in 0.5 ml buffer consisting of 10 mM Tris-HCl (pH
7.5), 125 mM NaCl, 1% Triton X-100, 1 mM PMSF, and a protease
inhibitor mixture (CompleteTM protease inhibitor mixture, Boehringer
Mannheim). Cell extracts were incubated on ice for 20 min and
centrifuged (13,000 g, 4°C, 20 min). The protein
concentration of the supernatant was determined in a Bradford protein
assay (13)
.
One hundred micrograms protein of each whole cell extract was boiled for 10 min in denaturating sample buffer consisting of 10% glycerol, 1% SDS, 1% ß-mercaptoethanol, 10 mM Tris-HCl (pH 6.8), and 0.01% bromphenol blue, then separated on 4–20% acrylamide gel and transferred on an Immuno-BlotTM PVDF Membrane (Bio-Rad). After blocking in 3% skim milk powder dissolved in PBS, the membrane was incubated for 90 min with 0.1 µg/ml PCNA, 0.1 µg/ml p53, 1 µg/ml Par-4, 2 µg/ml cytochrome c, 2 µg/ml Bax (Santa Cruz Biotechnology, Santa Cruz, Calif.), and 2 µg/ml Bcl-2 antibodies (Oncogene, Cambridge, Mass.). Subsequently, the blot was washed three times in 0.05% Tween 20 in PBS and incubated with 1:5,000 dilutions of goat anti-mouse or goat anti-rabbit conjugates (IgG-HRP, Pierce, Rockford, Ill.), respectively. After extensive washing in 0.05% Tween 20/PBS, a chemoluminescent substrate (SuperSignal®, Pierce), mixed with equal volumes of enhancer and luminol was added to the membrane, which was incubated for 5 min and exposed to the ECLTM HyperfilmTM (Amersham, Buckinghamshire, U.K.).
Caspase 3 activity
Caspase 3 activity was determined by cleavage of by the
biological substrate poly-(ADP-ribose) polymerase (PARP) from 116 kDa
into a 89 kDa fragment. Log-phase growing BPH-1 cells
(5x106) were either exposed to 1% SMP or UV
irradiated (1 kJ/m2). Nuclear extracts were
prepared after 6, 9, 12, 15, 18 and 21 h. Cells were trypsinized,
washed, and the cell pellet was resuspended in 135 µl lysis buffer
consisting of 100 mM Tris-HCl pH 7.3, 500 mM NaCl, 12 mM
2-mercaptoethanol, 25 mM
K2S2O5,
and 17% glycerin. After three freeze/thaw cycles, 45 µl protamine
sulfate (1%) was added and the extracts were centrifuged for 30 min
(10,000 g, 4°C). Nuclear extracts were desalted by
spinning through a Sephadex G25 column (5 min, 500 g).
Denaturating sample buffer was added to the desalted probes, which were
separated on 10% acrylamide gels. Western blot was performed as
described earlier except that an anti-PARP polyclonal rabbit IgG (0.5
µg/ml, Upstate Biotechnology, Lake Placid, N.Y.) was used.
To determine caspase activity, a caspase 3 colorimetric protease assay was performed. Log-phase growing BPH-1 cells (1x107) were lysed after 3, 6, and 9 h of incubation with 1% SMP in 200 µl buffer consisting of 50 mM HEPES/NaOH pH 7.4, 100 mM NaCl, 1% glycerol, 0.1% CHAPS (Sigma), 10 mM DTT, 0.1 mM EDTA. After sonification on ice, solubilized cells were centrifuged (15 min, 4°C, 10,000 g) and 90 µl cytosolic extract/well was incubated with 10 µl colorimetric caspase 3 substrate Ac-DEVD-pNA (Alexis Biochemicals, Läufelingen, Switzerland). After 1 h of incubation at 37°C, the OD of the 96-well microtiter plate was determined at 410 nm in the multiwell spectrophotometer.
cAMP [125I] assay system
BPH-1 cells (1x106) were starved for
24 h in serum-free medium, then stimulated for 3 h with
single HPLC fractions diluted 1:10 in RPMI 1640 (containing
1x10-4 M 3-isobutyl-1-methylxanthine) or with
1% of unfractionated SMP. Forskolin (50 µM), a nonspecific
stimulator of the adenylate cyclase, was used as positive control.
Supernatants were analyzed in duplicate for their content of cAMP in a
competitive RIA (Amersham Pharmacia Biotech). In brief, duplicates of
cAMP standard and samples were diluted in assay buffer (0.05 M acetate
buffer, 0.01% sodium azide) to a final volume of 500 µl. Acetylation
was performed by the addition of 25 µl acetylation reagent (1 vol
acetic anhydride and 2 vol triethylamine). Two aliquots (100 µl) were
transferred into the assay vials and incubated with 100 µl
[125I] cAMP tracer and 100 µl of antiserum
(rabbit anti-succinyl cAMP serum) at 4°C for 15–20 h. For bound/free
separation, 100 µl donkey anti-rabbit IgG coated to magnetizable
polymer particles was added, incubated for 10 min, then centrifuged and
counted in a gamma counter (1470 Wizard, Wallac, Sweden) for 1 min.
cAMP concentration of the samples were calculated after logit/log
transformation of the cAMP standard curve.
Tyrosine phosphorylation
BPH-1 cells (5x106) were starved in
serum-free RPMI 1640 for 24 h. Medium was changed to RPMI 1640
containing 1% SMP or HPLC fraction ‘50 min’ responsible for
apoptosis. After 5 or 10 min, plates were placed on ice and washed with
ice-cold PBS. Prechilled lysis buffer containing 20 mM HEPES pH 7.4, 2
mM EGTA, 50 mM ß-glycerophosphate, 1 mM DTT, 1 mM
Na3VO4, 1% Triton X-100,
10% glycerol, 2 µM leupeptin, 10 U/ml aprotinin, and 1 mM
phenylmethylsulfonyl fluoride was added and cells were scraped
carefully from the plate with a rubber policeman. Cells were lysed on
ice for at least 20 min. After centrifugation (10,000 g, 20
min, 4°C), the clear supernatant was harvested. Cell extracts were
subjected to electrophoresis (10% acrylamide gel) and Western blot
analysis was performed using an mAb directed against phosphotyrosine
(clone 4G10, Upstate Biotechnology). Instead of skim milk powder, 3%
BSA fraction V (Sigma) was used for blocking and mAb incubations. To
ensure equal protein loading and membrane transfer, blots were stripped
and reprobed with an mAb directed against the oncoprotein receptor
erbB2 (clone Ab-10, Neomarkers, Union City, Calif.), which is known
to be expressed in prostatic epithelial cells.
Terminal deoxynucleotidyl transferase (TdT) -mediated dUTP
nick-end labeling (TUNEL)
Log-phase growing BPH-1 cells (1x106)
were stained for apoptotic DNA fragmentation with the in
situ cell death detection kit (TUNEL) according the
manufacturer’s instructions (Boehringer Mannheim). Cells were
incubated for 24 and 48 h with 1% SMP or UV irradiated (1
kJ/m2). Subsequently they were harvested, washed
in PBS, fixed in 4% paraformaldehyde (30 min), and permeabilized in
0.1% Triton X-100/0.1% sodium citrate on ice (2 min). Thereafter,
1 x 105 cells were resuspended in 50 µl
TUNEL reaction mix containing FITC-dUTP and TdT, and incubated for 60
min at 37°C in a humidified chamber. The reaction was stopped by the
addition of 2 µl 0.5 M EDTA; after extensive washing in PBS, cells
were embedded and viewed on the confocal scanning system.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Serum-starved BPH-1 cells displaying a low proliferative
activity were resistant to SMP-induced cell death and did not show
significant reduction in cell viability within 24 h of incubation
(Fig. 1A
).
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A low molecular weight (LMW) factor of SMP is responsible for cell
death
Direct comparison of the unfractionated SMP with each fraction
revealed an interesting observation. Only fraction 50, eluted at 50 min
and corresponding to a calculated 2–4 kDa, had an effect on BPH-1 cell
viability similar to that seen with unfractionated SMP (Fig. 2A
). Most cells died within 20 h of incubation, when
doses of 1% were applied. This effect was not only specific for
immortalized BPH-1 cells, as the responsible fraction 50 induced cell
death in androgen-independent and -dependent prostate carcinoma cell
lines PC-3 and LNCaP as well as in primary cultures of human prostate
epithelial cells (Fig. 4)
. Boiling of SMP (10 min, 100°C) had no
influence on the activity of fraction 50. Proliferation assays were
performed with two important prostaglandins (PGE) present in human SMP.
Neither PGE-1 nor PGE-2 influenced BPH-1 cell growth and viability at
concentrations ranging from 10-5 to
10-8 M. Essentially the same was found for sex
steroid hormones, such as dihydrotestosterone and ß-estradiol, tested
at concentrations ranging from 10-6 to
10-9 M (data not shown). Thus, the low molecular
factor presumably belonged to neither steroid hormones nor
prostaglandins.
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Signal transduction pathway of LMW SMP
Signal transduction of fraction 50 min (2–4 kDa) was investigated
in comparison to unfractionated SMP. Activity of the adenylyl cyclase,
based on the production of cAMP, was measured after 3 h of
incubation with medium containing whole SMP or the purified single
fractions, respectively. As expected, unfractionated SMP was a potent
stimulator of the adenylyl cyclase. Compared to fractions 54 and 55,
containing prostaglandins in µM concentrations, fraction 50 increased
intracellular cAMP levels fivefold above constitutively produced cAMP
(Fig. 3A
). Tyrosine phosphorylation of membrane receptors and
intracellular signal transduction molecules were also investigated
(Fig. 3B
). Compared to untreated cells, incubation with
fraction 50 did not alter membrane or intracellular protein
phosphorylation within 10 min. In contrast, unfractionated SMP induced
a strong phosphorylation of proteins of 185 and 125 kDa. The
responsible factor was a protein with a molecular mass greater than 20
kDa (data not shown).
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Morphological changes of nucleus and cytoskeleton induced by SMP
Cell morphology changed rapidly when human prostate epithelial
cells were exposed to 2% SMP. Compared to untreated control cells,
shrinkage of cytoplasm could be observed within 6 h of incubation
(Fig. 4
), when cells also started to detach. This effect has been observed in
immortalized BPH-1 cells, prostate cancer cell lines (PC3 and LNCaP),
and primary cultures of human prostate epithelial cells (Fig. 4)
. It is
noteworthy that loss of filamentous actin and subsequent destruction of
the organized cytoskeleton occurred after 6 h in UV-irradiated as
well as in SMP-exposed prostate epithelial cells. Nuclear morphology of
BPH-1 cells changed significantly after SMP treatment, as shown by DAPI
staining of the nuclear DNA (Fig. 7A
). Compared to untreated
cells, incubation with SMP resulted in an increased condensation of
nuclei and in irregularities of nuclear shape and size. UV-irradiated
cells displayed a weaker condensation of their nuclei.
|
SMP-induced loss of membrane asymmetry
Loss of phospholipid asymmetry in the cell membrane was observed
in SMP-treated cells. Externalization of phosphatidylserine from the
inner to the outer site of the cell membrane was measured by binding of
FITC-conjugated annexin V. Dead cells with destroyed cell membrane
could be discriminated by double staining with propidium iodide. After
4 to 6 h, BPH-1 cells stained single positive for annexin V
(Fig. 5B
, lower right quadrant), indicating processes of membrane
destabilization. The same was observed in UV-irradiated cells (1
kJ/m2), where accumulation of early apoptotic
cells could be detected (Fig. 5A
, lower right quadrant).
SMP-treated cells did not accumulate in the early apoptotic status, but
migrated continuously in the late apoptotic/necrotic status and became
double positive (Fig. 5B
, upper right quadrant).
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SMP-induced DNA fragmentation
Nuclear fragmentation of BPH-1 cells was determined after 24 and
48 h of incubation with SMP (1%) and after UV irradiation (1
kJ/m2) by FACS analysis with propidium iodide or
TUNEL. Compared to proliferating BPH-1 cells, SMP was responsible for a
decrease of G2/M phase cell nuclei and condensation of G1 phase nuclei
after 24 h of incubation (Fig. 6
). Furthermore, G2/M phase cells disappeared after 48 h, and cells
in G1 phase showed a strong fragmentation of their DNA, as was observed
with UV-irradiated BPH-1 cells (Fig. 6)
. SMP-induced DNA fragmentation
was verified by TUNEL staining (Fig. 7B
). Compared to untreated control cells, SMP treatment of
BPH-1 cells for 48 h resulted in a TUNEL-positive staining of
30 ± 4%.
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SMP did not increase caspase 3 activity
Caspase 3 activity was proved either by cleavage of the natural
substrate PARP or by the synthetic colorimetric peptide Ac-DEVD-pNA.
Only UV irradiation resulted in increased caspase 3 activity, as
determined by PARP Western blots. The caspase 3-specific 89 kDa
fragment of PARP was clearly visible as early as 9 h after
irradiation (Fig. 8A
), which was not the case in SMP-treated BPH-1 cells. The
same results were obtained by measurement of caspase 3 activity with
the synthetic substrate in cytosolic extracts of BPH-1 cells. Caspase 3
rapidly became activated (2 h after UV irradiation) and reached maximum
activity after 4 h (Fig. 8B
). SMP treatment resulted in
a complete proteolytical breakdown of PARP within 21 h of
incubation, independent of caspase 3 activity (Fig. 8A
).
|
SMP-induced loss of mitochondrial potential and decreased Bcl-2
protein levels
Mitochondrial disintegration and loss of inner membrane potential
occurred rapidly after SMP treatment of BPH-1 cells, as determined by
staining with the mitochondrial potential sensitive dye JC-1. Exposure
to SMP primarily resulted in an activation of mitochondria and a high
oxidative potential, as indicated by strong red staining (Fig. 9B
). As a secondary event, mitochondria lost their potential
into the cytoplasm, as visualized by strong green staining of
organelles and cytoplasm (Fig. 9C
), followed by a loss of
staining due to mitochondrial disintegration processes (Fig. 9D
). These processes occurred within the first 6 h
after SMP contact, before dramatic changes of cellular and nuclear
morphology became visible. The proto-oncogene Bcl-2 protein levels
declined after SMP exposure and completely disappeared after 9 h
of incubation (Fig. 10B
). In parallel, a protein of 38 kDa, cross-reacting
with the Bcl-2 mAb, increased after 6 and 9 h of SMP treatment
whereas Bax levels remained nearly unaltered within 9 h of such
incubation.
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SMP induced proteolytical cleavage of p53 and increased expression
of the prostate apoptosis response gene (Par-4)
SMP exposure had an influence on the protein levels of the
proapoptotic gene product p53. Due to viral immortalization, BPH-1
cells permanently express p53, inactivated by the large T antigen of
SV40. Cellular p53 decreased immediately after SMP exposure and was
cleaved to a shorter fragment of 45 kDa after 9 h (Fig. 10A
). Moreover, SMP exposure led to an increase of Par-4
protein; compared to proliferating control cells, Par-4 protein
expression was up-regulated after 6 h and vanished after 9 h
(Fig. 10C
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Thus, apart from better pathological understanding,
preventative steps based on pharmacological therapeutics are necessary
increasingly. Aside from hormonal changes in the elderly men, it has
been assumed that BPH is caused by disturbances in apoptosis of stromal
cells of the prostate (15)
, which decreases with age,
although the proliferative index remains unaltered (16)
.
Furthermore, degenerated prostatic epithelial cells, which evade
apoptosis, accumulate mutations, become insensitive to further
anti-growth signals, and acquire limitless replicative potential,
forming the basis for neoplastic transformation and subsequent
development of prostatic cancer (17)
.
Growth of the prostate with age is a complex, poorly understood
process. In addition to the indispensable steroid, testosterone,
changes of endocrine factors, such as luteinizing hormone-releasing
hormone, growth hormone, IGF-I, and PRL, have been shown to influence
prostate growth homeostasis (3
, 5
, 6
, 18
, 19)
. Moreover,
local changes in the balance of growth-promoting and -inhibiting
factors seem to be important mediators for the development of BPH
(7)
.
Prostate epithelial cells produce substances beneficial for optimal
fertility, which might act in a retrograde manner on the epithelium by
influencing cell proliferation, differentiation, and
epithelial–stromal interactions. In this study, we describe the
isolation of a potent apoptosis-inducing factor from human seminal
fluid by HPLC. This molecule, presumably a small polypeptide with a
molecular mass of 3.5 kDa, superimposed all proliferative effects of
other growth-promoting factors present in human SMP. Thus,
unfractionated SMP did not enhance BPH-1 cell proliferation, but cells
lost viability within 24 h under serum stimulation (Fig. 1B
). ‘Programmed cell death’ was determined by nuclear DNA
fragmentation (Fig. 6)
and early loss of phospholipid membrane
asymmetry (Fig. 5)
. Apoptosis could be induced with the SMP samples of
10 of 12 probands. Thus, the responsible substance must be present in
the SMP of most males. Compared to the apoptosis-inducing SMP samples
and the common SMP pool, these two patients had an unusual protein
pattern and significant lower amounts of fraction 50 as determined by
HPLC analysis (data not shown). Serum-starved BPH-1 cells displaying a
very low proliferative activity responded to increasing concentrations
of SMP with no significant loss of cell viability (Fig. 1A
),
excluding the possibility that the substance might belong to bacterial
endotoxins.
Signal transduction of fraction 50 was mediated primarily by activation
of seven-transmembrane receptor molecules, stimulation of the adenylyl
cyclase, and production of cAMP (Fig. 3A
). Many short
peptides—up to 50 amino acid residues present in prostate and SMP,
i.e., thyrotropin-releasing hormone-related polypeptides
(20)
—are known to exert their actions via stimulation of
adenylyl cyclases. Presumably this cAMP-mediated signal pathway led to
apoptosis, since no predominant tyrosine phosphorylation of membrane
receptors or associated protein kinases could be observed within 10 min
after stimulation (Fig. 3B
). The involvement of cAMP in
apoptosis is not very surprising, as cAMP-dependent protein kinases
have been shown to regulate apoptosis by phosphorylation of Bcl-2 and
reducing Bcl-2/Bax dimerization (21)
. Elucidation of the
SMP-triggered apoptotic mechanism revealed common processes, but also
differences to the classic apoptotic stimulus, UV irradiation. Nuclear
fragmentation (Fig. 6)
and generation of DNA breaks (Fig. 7B
) were quite similar in UV-irradiated and SMP-treated
BPH-1 cells, and early apoptotic translocation of phosphatidylserine in
the cell membrane (Fig. 5)
and disintegration of filamentous actin
cytoskeleton occurred within 6 h. In both apoptotic processes,
TUNEL-positive cells, characteristic of late apoptosis and of increased
DNA strand breaks and fragmentation were observed (Fig. 7B
).
Most noteworthy, activation of caspase 3 and cleavage of PARP, a
nuclear DNA repair protein characteristic of UV irradiation-induced
apoptosis, was not detected in cells exposed to SMP (Fig. 8A
, B
). Thus, the observed proteolytic breakdown of PARP between 18
and 21 h must be due to other caspases or proteases. SMP induced
disintegration of mitochondria and activated mitochondrial-triggered
apoptotic processes. A rapid loss of mitochondrial potential could be
determined by the mitochondrial potential sensitive dye-JC-1 (Fig. 9A
, B
, C
, D
). Further, a decline in mitochondrial Bcl-2 protein
levels was observed that disappeared completely after 9 h of
incubation, whereas Bax protein levels remained unaltered (Fig. 10B
). Thus, changes in the Bax/Bcl-2 ratio and in the
mitochondrial rheostat of cell death and survival could be responsible
for induction of apoptosis, as demonstrated with rodent prostate
epithelial cells after castration (22)
. Decreases of Bcl-2
protein levels have been shown to favor the formation of Bax/Bax
homodimers, which in turn interact with the adenine nucleotide
translocator and activate the mitochondrial permeability transition
pore complex (23)
. Recently, it has been demonstrated that
overexpression of Bax-like proteins or their enforced dimerization
kills mammalian cells by provoking DNA condensation and membrane
alterations without involvement of caspase activation
(24)
. This observation could explain why SMP treatment
induced apoptosis without the involvement of the key caspase, caspase
3. Presumably, alterations in the relative concentrations of
mitochondrial Bcl-2 pro- and antiapoptotic family members were
sufficient to induce apoptosis.
SMP treatment resulted in an immediate increase of cellular p53 protein
levels after 3 h of incubation, which might be a consequence of
increased protein stability or reduced ubiquitinylation and
degradation. In the initial stages of apoptosis, high p53 levels might
be important to prevent further cell divisions, provoke growth arrest,
and ensure DNA repair. In later stages of apoptosis, p53 was cleaved
(Fig. 10A
); at this point, inhibition of DNA repair by
cleavage of p53 might be necessary to ensure the completion of
apoptosis. Proteolytic cleavage of p53 has been reported to be
important for the activation of this molecule in response to DNA damage
and for interaction with single-stranded DNA (25)
. Since
BPH-1 cells are SV 40 large T immortalized, SMP-induced changes in p53
protein were also analyzed in nonimmortalized primary cultures of human
prostate epithelial cells. Again, after an initial increase, p53
protein degradation occurred within 6–9 h (data not shown). Apart from
p53, prostate apoptosis response 4 gene (Par-4) protein levels were
elevated in early SMP-induced apoptosis. Par-4, a leucin zipper protein
with nuclear localization sequence, has been discovered to be a
differentially regulated gene in androgen-dependent apoptosis of rodent
prostate epithelial cells (26)
. Moreover, Par-4 has been
shown to be up-regulated in an apoptosis-specific manner and not to be
induced by effectors of growth stimulation, oxidative stress, growth
arrest, or necrosis (27)
. Most significant, protein levels
of Par-4 could only be detected after 6 h in the cytoplasm of
SMP-treated cells, whereas it vanished again after 9 h, possibly
due to apoptosis-specific gene expression in the first stages of
apoptosis followed by rapid degradation (Fig. 10C
).
Better characterization of the apoptosis-inducing 2–4 kDa fraction of human SMP remains to be achieved. This fraction contains a potent inducer of apoptosis acting primarily on proliferating cells, as shown for nonimmortalized and immortalized human prostate epithelial cells and for prostatic cancer cell lines PC-3 and LNCaP. This molecule could be physiologically important either for the elimination of old degenerated secretory epithelial cells or for differentiation processes occurring in the permanent renewal of prostatic epithelium. Additional studies will provide insight as to how this factor influences renewal and secretory function of the prostate epithelium, and elucidate whether changes in its concentration contribute to the development of BPH or prostatic cancer.
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ACKNOWLEDGMENTS |
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Received for publication May 22, 2000.
Revision received August 30, 2000.
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REFERENCES |
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