Mitogenic Effect of Nerve Growth Factor (NGF) in LNCaP Prostate Adenocarcinoma Cells: Role of the High- and Low-Affinity NGF Receptors
Maria Angela Sortino,
Fabrizio Condorelli,
Carlo Vancheri,
Andrea Chiarenza,
Renato Bernardini,
Ugo Consoli and
Pier Luigi Canonico
Institutes of Pharmacology (M.A.S., F.C., A.C., R.B.), Respiratory
Diseases (C.V.), and Hematology (U.C.) University of Catania School
of Medicine 95125 Catania, Italy
Department of Internal
Medicine (P.L.C.) Section of Pharmacology University of Pavia
School of Medicine 27100 Pavia, Italy
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ABSTRACT
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We have investigated the effect of nerve growth
factor (NGF) in the androgen-dependent, prostate adenocarcinoma LNCaP
cell line. Exposure of LNCaP cells to NGF resulted in a significant
increase of cell proliferation. The effect was concentration dependent
and equally present in serum- or charcoal-stripped
serum-supplemented and serum-deprived conditions. The mitogenic action
of NGF was accompanied by an enhanced expression of prostate-specific
antigen (PSA) and resulted additive to the proliferative effect of
dihydrotestosterone. The proliferative effect of NGF appeared to be
mediated by the high-affinity NGF receptor,
p140trka. Only
p140trka, but not the low-affinity NGF
receptor, p75LNGFR, was expressed in LNCaP
cells; both the proliferative response and the phosphorylation of
p140trka upon NGF treatment were prevented by
the tyrosine kinase inhibitor K252a. LNCaP cells transiently
transfected with the cDNA encoding for p75LNGFR
appeared more sensitive to NGF, as demonstrated by the increased number
of p75LNGFR-transfected LNCaP cells exposed for
72 h to NGF compared with wild LNCaP cultures. However,
p75LNGFR-transfected LNCaP cells rapidly
underwent apoptotic death when deprived of NGF. Our study demonstrates
the physiological relevance of NGF in the regulation of prostate cell
proliferation and the relative contribution of the high- and
low-affinity NGF receptors in this control.
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INTRODUCTION
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Paracrine and autocrine mechanisms play a major role in the
control of normal and aberrant growth of the prostate (1 2 ). In this
respect, growth factors and their receptors seem to be primarily
involved (3 ). Thus, insulin-like growth factor-I (IGF-I), epidermal
growth factor, and transforming growth factor-
exert their
biological effects on prostate epithelium being produced by stromal
and/or epithelial cells (4 5 6 7 ). Peptides of the fibroblast growth
factor (FGF) family are also considered essential factors for prostate
proliferation (8 9 ), and changes in the expression of FGF and/or FGF
receptors positively correlate with the malignant progression and
metastatic potential of several neoplasias (10 11 ), including prostate
tumors (12 13 ).
Nerve growth factor (NGF) has been identified in human prostate (14 15 ) where it localizes to the stromal compartment (16 ). The modulatory
action of a NGF-like protein released by prostatic stromal cells on the
proliferation of human prostatic epithelial cells (17 ) clearly
indicates a paracrine function for NGF in the control of prostatic
growth.
NGF exerts its action through stimulation of a high-affinity receptor,
identified as a member of the tyrosine kinase receptor family, termed
p140trka, and a low-affinity high-capacity
transmembrane receptor, named p75LNGFR, whose
transduction signal has not been completely elucidated yet (all
reviewed in Ref. 18 ). p75LNGFR is expressed in
prostate epithelium in vivo (16 ) and in primary cultures of
normal prostate epithelial cells (19 ), but it is absent from prostate
stroma in vivo (16 ) and from a human prostatic stromal cell
line (19 ). More interestingly, in benign prostate hyperplasia and in
prostatic adenocarcinoma, p75LNGFR expression is
reduced or absent (19 20 ), and it is completely missing in prostate
metastatic cell lines (19 ), suggesting an inverse association of
p75LNGFR expression and the neoplastic
progression of human prostate.
The human prostatic adenocarcinoma LNCaP cells, obtained from a
supraclavicular lymph node metastasis (21 ), represent one of the
metastatic prostate tumor cell lines that do not express
p75LNGFR (19 ). LNCaP cells are largely used for
the study of prostatic cancer biology as they maintain several features
of human prostatic carcinoma including expression of high affinity
androgen receptors and production of prostate-specific antigen (PSA), a
marker of cellular differentiation in prostatic epithelial cells (21 ).
By using the LNCaP cell line, we have tried to characterize the
biological response of NGF in prostatic adenocarcinoma by focusing our
attention on the relative contributions of high- and low-affinity NGF
receptors in this action.
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RESULTS
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NGF Affects LNCaP Cell Proliferation
Exposure of LNCaP cells to NGF resulted in a significant increase
of cell proliferation. Interestingly, the entity of the stimulatory
effect produced by NGF was comparable to that induced by
dihydrotestosterone (DHT; 10 nM; Table 1
) and was equally present in cells
cultured in the presence or in the absence of serum (Table 1
) or in
charcoal-stripped serum (CSS)-containing culture media (not shown). As
expected, stimulation of LNCaP proliferation by DHT was attenuated in
the presence of serum (Table 1
). Because of the androgen dependence of
LNCaP cells for their cellular growth and of the similar response
observed in different culture conditions, CSS-supplemented medium was
used in all subsequent experiments. A first significant stimulatory
effect on LNCaP cell proliferation by NGF was observed at a
concentration of 3 ng/ml, as assessed by either cell counting and
[3H]thymidine incorporation (Fig. 1
, upper panel),
and was maximal in a range of concentrations of 1025 ng/ml. This
proliferative effect of NGF exhibited also time dependency, as it was
already present after a single treatment with NGF (48 h), was more
pronounced after a repeated stimulation (7 days), and maximal after a
much more prolonged exposure to the growth factor (2 week-treatment;
Fig. 1
, lower panel). Under these conditions no apparent
change of LNCaP cell morphology and phenotype was evident by phase
contrast microscopy observation (data not shown). The stimulatory
action of NGF on LNCaP cell growth was not unique as other growth
factors including basic FGF and IGF-I were also able to stimulate LNCaP
cell proliferation (Table 2
). Although,
as mentioned above, treatment with NGF and DHT modified LNCaP cell
proliferation to a similar extent, the mechanisms involved in their
action seem to be completely independent as shown by the additive
effect on LNCaP cell proliferation observed after repeated, combined
addition of increasing concentrations of DHT and NGF (Fig. 2
).
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Table 1. Effect of NGF and DHT on the Proliferation of
LNCaP Cells Grown in Serum-Supplemented and Serum-Deprived
Conditions
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Figure 1. Concentration-Response and Time Course Curves of
NGF Effect on LNCaP Cell Proliferation
LNCaP cells maintained in CSS were exposed to increasing concentrations
of NGF for 8 days (upper panel). Cells were then either
harvested and counted at the hemocytometer or incubated with 1 µCi/ml
[3H]methylthymidine during the last 6 h of
incubation and evaluated at a scintillation counter after precipitation
of the acid-insoluble cellular fraction. The lower panel
shows the effect of different exposure times to 25 ng/ml NGF on LNCaP
cell growth as evaluated by cell counting. All data are mean ±
SE from three to five independent studies performed in
triplicate. *, P < 0.01 vs.
respective control.
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Figure 2. Additive Effect of DHT and NGF in Stimulating LNCaP
Cell Proliferation
CSS-cultured LNCaP cells were repeatedly treated with NGF (25 ng/ml)
and increasing concentrations of DHT (10 nM) for 15 days
before detachment with trypsin and counting at the hemocytometer. Data
are mean ± SE of four independent studies.
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Effect of NGF on the Expression of PSA
Continuous exposure of LNCaP cells to NGF markedly enhanced the
expression of PSA, a typical marker of cellular differentiation in
prostatic epithelial cells. Increased immunostaining for PSA was
observed after exposure of LNCaP cells to NGF as assessed by the
percentage of immunopositive cells (Fig. 3
). Treatment with DHT produced a similar
stimulatory effect on the production of PSA (Fig. 3
). Western blot
analysis revealed a single band with a molecular mass of approximately
30 kDa that was clearly enhanced after treatment of LNCaP cells with
either NGF or DHT (Fig. 3
).

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Figure 3. NGF Treatment Increases the Expression of PSA in
LNCaP Cells
LNCaP cells exposed for 7 days to either vehicle, NGF (25 ng/ml), or
DHT (10 nM) were fixed with 4% paraformaldehyde for
immunocytochemistry or lysed and processed for protein extraction and
Western blot analysis. An antihuman anti-PSA antibody raised in rabbits
was used. On the left, positive PSA immunostaining in
NGF- (middle) and DHT-treated (lower
panel) cultures, compared with control (upper
panel), was visualized with diaminobenzidine. Protein bands (on
the right) were visualized by chemiluminescence. The
same membrane was probed with a polyclonal anti-ß-tubulin antibody
raised in goat (1:200) to control for protein loading.
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The High-Affinity NGF Receptor p140trka
Mediates the Stimulatory Action of NGF on LNCaP Cell Growth
To analyze in more detail the effect of NGF on LNCaP cells, the
expression of high- and low-affinity receptor proteins for NGF was
examined. LNCaP cells expressed the high-affinity NGF receptor
p140trka (Fig. 4
),
but not the low-affinity p75LNGFR (not shown) as
revealed by immunocytochemistry. In addition, the presence of
p140trka in LNCaP cells was validated by
cytofluorimetric analysis that revealed more than 97% LNCaP cells
positive for p140trka. Expression of
p140trka in LNCaP cells and its phosphorylation
upon NGF treatment were further confirmed by Western blot analysis
(Fig. 5
). On a functional basis, the
involvement of p140trka in NGF action was
suggested by complete prevention of the stimulatory effect on LNCaP
cell growth by treatment with the tyrosine kinase inhibitor K252a (Fig. 5
) that, at the concentration used (1 nM),
exhibits relative specificity for trka (22 ). In addition, in
the presence of K252a, the enhanced tyrosine phosphorylation induced by
receptor activation by NGF was completely prevented (Fig. 5
).

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Figure 4. LNCaP Cells Show Positive Immunostaining for the
High-Affinity NGF Receptor p140trka
LNCaP cells were fixed with 4% paraformaldehyde, exposed to
Triton-X100 for 5 min at 4 C, and analyzed by immunocytochemistry after
labeling with a goat antihuman p140trka (1
µg/ml) followed by a biotinylated anti-rabbit IgG (1:200). A
culture in which incubation with the primary antibody was omitted is
shown for comparison (lower panel).
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Figure 5. Inhibitory Action of K252a on NGF-Stimulated LNCaP
Cell Proliferation and Tyrosine Phosphorylation of
p140trka
A, LNCaP cells were exposed for 48 h to K252a (1
nM) either alone or in combination with NGF (25
ng/ml). Cells were counted with the aid of a hemocytometer. Data are
mean ± SE of four independent studies performed
in triplicate. *, P < 0.05 vs. control;
**, P < 0.05 vs.
NGF-treated. B, Expression and tyrosine phosphorylation of
p140trka in basal conditions (lanes 1 and 5) and
after treatment with NGF (lanes 2 and 6), K252 (lanes 3 and 7), and NGF
plus K252 (lanes 4 and 8). Cell lysates were immunoprecipitated with
the polyclonal anti-p140trka antibody. Membranes
were immunoblotted with either the polyclonal
anti-p140trka antibody (lanes 14) or a
monoclonal antiphosphotyrosine antibody ( PY; lanes 58).
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Evaluation of Cellular Growth and Viability in
P75LGFNR-Transfected LNCaP Cells
To control for p75LNGFR membrane expression
in p75LNGFR-transfected LNCaP cells,
cytofluorometric analysis was performed by indirect immunofluorescence
with a monoclonal antibody recognizing p75LNGFR.
The efficacy and specificity of the anti-p75LNGFR
antibody were evaluated in parallel cultures of differentiated SK-N-BE
human neuroblastoma cells expressing p75LNGFR
(not shown). Figure 6
depicts the shift
in immunofluorescence intensity in
p75LNGFR-transfected (solid histogram)
vs. vector-transfected LNCaP cells. Immuno-4 processing
(Coulter Electronics, Hialeah, FL) was applied to calculate the
percentage of positive cells (40.6 ± 2.5%). Expression of
p75LNGFR in
p75LNGFR-transfected cells was confirmed by
immunocytochemistry that revealed a subpopulation of cells positively
stained by the anti-p75LNGFR antibody (Fig. 6
, right photomicrograph). To assess the proliferation rate of
LNCaP cells expressing p75LNGFR, cells were grown
in 24-well multiwell plates, and incubated with NGF from day 1 after
transfection for 72 h. NGF (25 ng/ml) was added daily. This
treatment produced an increase in cell proliferation that was 7075%
over control (Fig. 7
) and exhibited time
dependency as cells deprived of the growth factor 1 day before
harvesting arrested their proliferation rate (Fig. 7
). The 24-h
wash-out period was chosen to be sure that
p75LNGFR was absolutely free of ligand due to the
specific kinetics of binding of NGF to this receptor, which exhibits
very fast association and dissociation rates (18 ). The stimulatory
effect on cell growth induced by NGF (25 ng/ml) was more pronounced
(~35% over that induced in untransfected cells) in
p75LNGFR-transfected LNCaP cells (Fig. 7
).
However, this potentiation was evident only at this maximally effective
concentration of NGF. In fact, treatment of
p75LNGFR-transfected LNCaP cells with submaximal
concentrations of NGF (between 1 and 10 ng/ml) was unable to produce a
further increase in cell proliferation when compared with untransfected
cultures (not shown). Interestingly, complete removal of NGF from the
transfected cell culture during the last 24 h of incubation
resulted in a final cellular density that was below that observed in
untreated p75LNGFR-transfected LNCaP cells (Fig. 7
). No significant change of growth rate could be detected in
p75LNGFR-transfected LNCaP cells when not exposed
to NGF (not shown).

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Figure 6. Flow Cytometric and Immunocytochemical Analysis of
p75LNGFR Expression in p75LNGFR-Transfected
LNCaP Cells
LNCaP cells transfected with the pCEP4 plasmid containing the human
p75LNGFR cDNA (top, solid histogram) or the
vector alone (open histogram) were detached from the
dish and immediately incubated with mouse antihuman
p75LNGFR (10 µg/ml) for 1 h followed by incubation
with FITC-conjugated goat antimouse IgG (1:100) for 45 min. Cells were
analyzed with a flow cytometer. At the bottom,
photomicrographs showing three p75LNGFR immunopositive
cells (right) in p75LNGFR-transfected
cultures. The lack of staining when primary antibody was omitted is
shown for comparison (left). Data shown are
representative of several independent experiments.
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Figure 7. Differential Response to NGF and NGF Withdrawal in
Wild (LNCaP) and p75LNGFR-Transfected
(LNCaPp75) LNCaP cells
LNCaP (open bars) and LNCaPp75
(cross-hatched bars) cells were treated with NGF (25
ng/ml, added daily) for 48 and 72 h or exposed to NGF for 48
h before deprivation for 24 additional h (wash-out period; NGF + w/o).
Data are mean ± SE of one experiment
representative of four, each run in triplicate.*, P
< 0.01 vs. respective control; ,
P < 0.05 vs. corresponding LNCaP
untrasfected cells.
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Apoptotic death in p75LNGFR-transfected LNCaP
cells was first evaluated by assessing fluorescein isothiocyanate
(FITC)-conjugated annexin V binding to phosphatidylserine
externalized on the cell surface. This specific method to evaluate
apoptotic death was chosen to make sure to detect early apoptotic
events. Immunofluorescence analysis at the flow cytometer revealed the
appearance of a distinct cell population, corresponding to about 30%
of the total cell population examined, that was immunopositive for
annexin V only in p75LNGFR-transfected LNCaP
cells deprived of NGF for 24 h after a 48 h treatment with
the growth factor (Fig. 8
, upper
panel). Conversely, minimal levels of annexin V binding (< 4%)
were detectable in NGF-treated
p75LNGFR-transfected LNCaP cells and either
NGF-treated or NGF-deprived untransfected LNCaP cells (Fig. 8
,
upper panel). Similar results were obtained by evaluating
apoptosis by assessment of the presence of a hypodiploid cell
population in propidium iodide-labeled cells (Fig. 8
, lower
panel).

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Figure 8. Detection of Apoptotic Cell Death in
p75LNGFR-Transfected LNCaP Cells by Flow Cytometric
Analysis
LNCaP and p75LNGFR-transfected LNCaP cells were either
continuously exposed to NGF (25 ng/ml) for 72 h with daily
addition (NGF) or treated with NGF and then deprived of the growth
factor during the last 24 h of incubation (NGF-w/o). Cells were
then detached and incubated with annexin V-FITC (1 µg/ml) or
propidium iodide before analysis by flow cytometry. The appearance of a
clear annexin V-positive cell population was evident only in
NGF-deprived p75LNGFR-transfected LNCaP cells (upper
panel). The intensity of fluorescence is plotted against cell
distribution. Similarly, a distinct hypodiploid cell population, as
detected by propidium iodide staining, appeared only in NGF-deprived
p75LNGFR-transfected LNCaP cells. Percent values refer to
the entity of the apoptotic population (lower panel).
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DISCUSSION
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The mechanisms underlying prostate tumoral growth are
still poorly understood. The interaction between stromal and epithelial
compartments within the gland and the involvement of paracrine and
autocrine events regulated by growth factors and their receptors seem
to play a major role in this regard (3 ). Since the early work
demonstrating the presence of NGF-like proteins in the prostate gland
of the guinea pig (14 ), a large body of evidence has indicated a
prominent role for NGF in the regulation of prostate growth. In
particular, the discovery that an NGF-like protein secreted by prostate
stromal cells stimulated growth of a prostatic epithelial tumor cell
line (17 ) allowed a more precise understanding of the effects mediated
by NGF at the prostate. Our data obtained in the prostatic
adenocarcinoma LNCaP cells indicate that NGF exerts a mitogenic action
in human prostate epithelial cells. Although better known for the
differentiating activity exerted in nerve cells (23 ), interaction of
NGF, as well as of other neurotrophins, with its specific receptors is
also able to elicit proliferation phenomena (24 25 26 ). These have been
observed in several cellular systems including undifferentiated
hematopoietic cells (27 ), normal human keratinocytes (28 ), a pancreatic
carcinoid (29 ), and a thyroid epithelial (30 ) cell line.
The stimulatory action induced by NGF on LNCaP cell growth was
concentration- and time-dependent; the entity of the effect produced
was comparable to that induced by basic FGF, but greater than that of
IGF-I probably because of the autocrine regulation of LNCaP cell
proliferation by this growth factor (31 ). In addition, the stimulatory
action on LNCaP cell growth was not influenced by culture conditions as
it was equally effective in the presence or absence of serum in the
culture media or when only steroids were removed from the incubation
buffer by the use of CSS. In line with this, stimulation of cell
proliferation by NGF has been reported in androgen-independent prostate
carcinoma cell lines (32 33 ). Furthermore, cotreatment of LNCaP cells
with NGF and DHT produced an additive effect, suggesting that the two
agents acted in parallel, through activation of completely independent
intracellular mechanisms.
Similarly to what was observed after treatment with DHT, a long-term
exposure of LNCaP cells to NGF was also able to induce increased
expression of the cellular differentiation marker, PSA. Androgens have
been reported to stimulate LNCaP cell proliferation at low
concentrations and to promote the production of PSA at high
concentrations (34 ). In the present study, the concentration-response
curve of DHT on LNCaP cell growth was shifted to the right compared
with that previously reported (34 ), a phenomenon that can be ascribed
to the batch of LNCaP cells used or to the different culture conditions
in which experiments were carried out. In our hands, however, 10
nM DHT, a concentration that was fully effective in
inducing increased LNCaP cell proliferation, was also able to enhance
PSA expression. Accordingly, NGF, at maximally effective concentrations
in promoting LNCaP cell growth, was also able to enhance PSA
production, suggesting that, in prostate adenocarcinoma cells, NGF can
maintain, at least in part, its differentiating properties while
inducing enhanced proliferation. Such a combined action of NGF has
already been reported in other cellular systems (35 ). The effect of NGF
on LNCaP cells, however, was not accompanied by any major change of
cellular phenotype or cytostructural modification. Moreover, although
other growth factors able to modulate LNCaP cell growth are known to
control proliferation and differentiation events in this cell line
inversely (36 ), the regulation of these phenomena appears extremely
complex as it involves a series of interactions between the
androgen-regulatory system and the growth factor-regulatory system that
are likely to take place at multiple intracellular levels in prostatic
cells.
NGF is known to exert its action through two distinct receptors: a
high-affinity tyrosine kinase receptor,
p140trka, and a low-affinity binding protein,
p75LNGFR (reviewed in Ref. 18 ). Although
p75LNGFR and p140trka are
coexpressed in many NGF-responsive cell types, independent expression
of individual receptors has also been observed. The ratio of the two
receptors seems to be crucial for the functional response elicited by
NGF, and coexpression of p75LNGFR and
p140trka is known to contribute to the formation
of a high-affinity binding site (18 ).
Prostatic LNCaP cells expressed p140trka, but
not p75LNGFR, as detected by immunocytochemistry,
cytofluorimetric, and Western blot analysis. The lack of
p75LNGFR in LNCaP cells was not surprising as a
progressive loss of the low-affinity NGF receptor from normal prostate
to adenocarcinoma and metastatic prostate tissue has already been
described (19 ). Accordingly, the mitogenic effect triggered by NGF in
LNCaP cells appeared to be mediated exclusively by activation of the
p140trka as demonstrated by the blockade of the
stimulatory action of NGF by the tyrosine kinase inhibitor K252a,
similarly to that already observed in BON pancreatic carcinoid cells
(29 ) and human keratinocytes (28 ). In strong support of a specific
involvement of p140trka in NGF-induced cell
proliferation, treatment with K252a was able to completely prevent the
phosphorylation at tyrosine residues of p140trka
observed upon NGF stimulation. However, the involvement of
p140trka in the regulation of cellular growth is
much more complex, as activation of the high-affinity NGF receptor is
also known to mediate growth arrest and differentiation not only in
neuronal cells such as neuroblastoma (37 ) and PC12 cells (38 ), but also
in NIH-3T3 fibroblasts, where NGF can elicit growth arrest by induction
of a cyclin-dependent kinase inhibitor (39 ), in various tumors of
neuroendocrine origin (reviewed in Ref. 40 ) and in human small cell
lung carcinoma cell lines (41 ).
p75LNGFR has recently been included in the
superfamily of death domain receptors, which comprises receptors for
some cytokines and several surface antigens (reviewed in Ref. 42 ). The
role of this p75LNGFR in the regulation of cell
death phenomena, however, is still controversial: intrinsic
p75LNGFR activity, as opposed to ligand-dependent
receptor effects, have in fact been described and they may
alternatively initiate proapoptotic or antiapoptotic signals (43 ). In
murine dorsal root ganglion neurons, p75NGFR
seems to mediate cell survival at very early times of development
whereas, at later stages, it triggers an intrinsic proapoptotic signal
that is completely prevented by NGF (44 ). Similarly, in immortalized
neural cells, NGF or an anti-NGF antibody abolish the death signal
mediated by unbound p75LNGFR (45 ). On the other
hand, however, NGF is also able to cause apoptotic death after binding
to its low-affinity receptor (46 ).
In nonneuronal cells, the exact role of p75LNGFR
has been much less investigated: the preferential involvement of
p140trka in the proliferating response of
tumoral, NGF-responsive cells (27 28 29 ) leads to the hypothesis that the
low-affinity NGF receptor might play a counterbalancing role in the
control of cellular growth. In LNCaP cells, however, expression of
p75LNGFR determines cooperation with
p140trka, as suggested by the greater mitogenic
effect observed in response to maximally effective concentrations of
exogenously added NGF. The lack of potentiation at lower NGF
concentrations, however, rules out possible changes in the affinity of
NGF for p140trka but rather suggests potential
interactions of p140trka- and
p75LNGFR-mediated signaling at a postreceptor
level. p75LNGFR seems to be endowed with
intrinsic activity that is unmasked only after exposure to NGF, as
revealed by the decreased cell number and the appearance of apoptotic
cell death after withdrawal of the growth factor. The lack of apoptosis
in response to NGF withdrawal in untransfected LNCaP cells excludes the
possibility that LNCaP cells become dependent on NGF for their survival
after a prolonged treatment with the growth factor. Partially in
contrast with our results, Pflug and Djiakiew (32 ) reported a
progressive reduced proliferation rate of TSU-prl prostatic epithelial
cells expressing low, intermediate, or high levels of
p75LNGFR in addition to the occurrence of
apoptosis upon NGF removal (32 ). The loss of
p75LNGFR expression in prostate adenocarcinoma
cells may thus represent a self-regulatory mechanism through which
proliferating cells control their growth processes. In line with this,
expression of p75LNGFR in human melanoma cells is
positively correlated with their chemoinvasion potential (47 ). On the
other hand, however, the ability of NGF deprivation to trigger
apoptotic events in p75LNGFR-transfected LNCaP
cells suggests a pivotal role for this receptor in the complex
regulation of proliferation phenomena by NGF.
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MATERIALS AND METHODS
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LNCaP Cell Culture
The androgen-sensitive human prostatic adenocarcinoma LNCaP
cells (obtained from American Type Culture Collection,
Manassas, VA), were maintained in RPMI-1640 medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 5% FCS,
and penicillin (100 U/ml)/streptomycin (100 µg/ml) (all from
Life Technologies, Inc.). In most studies, as specifically
indicated, FCS was replaced with CSS, obtained by treatment with
dextran-coated charcoal, to remove any steroid contaminant.
Cell Proliferation Studies
LNCaP cells plated at low density were cultured in FCS-,
CSS-supplemented, or serum-deprived media as specified in detail. Human
recombinant NGF (kindly provided from Genentech, Inc.,
South San Francisco, CA), the other growth factors and DHT
(Sigma, St. Louis, MO) were added every other day.
Cellular growth was assessed by total cell counts at the end of the
treatment period and by the rate of
[3H]thymidine incorporation in a 6-h labeling
period. For cell counting experiments, LNCaP cells, plated in 24-well
multiwell plates, were detached using a 0.25% trypsin/0.2% EDTA
solution (Life Technologies, Inc.) and counted with a
hemocytometer. Cell viability was determined by the trypan blue
(Sigma) exclusion test. For
[3H]thymidine incorporation studies, cells were
incubated with 21 µCi/ml [3H]methylthymidine
(New England Nuclear, Milan, Italy; specific activity, 20 Ci/mmol) for
6 h before precipitation with 1 N
HClO4. Incorporated radioactivity was quantitated
by liquid scintillation counting.
Immunocytochemistry
LNCaP cells were stained for the high-affinity NGF receptor
p140trka using a polyclonal anti-trk antibody
raised in rabbits (1 µg/ml; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). A monoclonal antihuman
p75LNGFR (10 µg/ml; Roche Molecular Biochemicals, Mannheim, Germany) was used to detect
p75LNGFR immunostaining in LNCaP cells.
Immunopositivity for PSA was assessed by using a polyclonal anti-PSA
antibody raised in rabbits (4 µg/ml; DAKO Corp.,
Glostzup, Denmark). Preparation of cells for immunostaining was
performed by fixing cells for 30 min with 4% paraformaldehyde. After
permeabilization in 0.1% Triton X-100 (Sigma) for 10 min
at room temperature (this step was omitted when looking for
p75LNGFR staining because the antibody used
recognizes an extracellular epitope of the receptor), cells were
repeatedly washed with phosphate buffer and exposed to the primary
antibody at 4 C overnight followed by incubation with biotinylated
antirabbit (or antimouse) IgG for 1 h. After reaction with
avidin-biotin-horseradish peroxidase (Vectastain ABC-Elite
kit, Vector Laboratories, Inc., Burlingame, CA), staining
was developed by exposure to 0.05% diaminobenzidine/0.01%
H2O2 for 10 min.
Western Blot Analysis
LNCaP cells plated in 100-mm plates were harvested at 4 C with a
lysis buffer containing phenylmethylsulfonylfluoride, aprotinin,
pepstatin A, and leupeptin. After centrifugation at 15,000 x
g at 4 C, the supernatant was processed for protein
concentrations according to the method of Bradford (48 ). Samples were
diluted in sample buffer and boiled for 5 min. Electrophoresis was
performed in 10% SDS-PAGE (30 mA/h) using 20 µg of total protein per
lane. After separation, proteins were transferred onto a nitrocellulose
membrane (Hybond ECL, Amersham Pharmacia Biotech Italia,
Milan, Italy), for 45 min at room temperature using a transblot semidry
transfer cell. After blocking, the membrane was incubated with anti-PSA
antibody (6.4 µg/ml) for 2 h at room temperature, and then
repeatedly washed and exposed to horseradish peroxidase-conjugated
antirabbit IgG for 1 h at room temperature. Proteins were
visualized using the enhancing chemiluminescence detection system (ECL,
Amersham Pharmacia Biotech, Arlington Heights, IL). The
same procedure was used for the Western blot analysis of
p140trka protein; to detect the phosphorylation
status of the receptor, proteins were incubated overnight at 4 C with
anti-p140trka. Immunocomplexes were precipitated
with antirabbit IgG-agarose beads for 1 h at 4 C before elution,
electrophoresis separation, and blotting with
anti-p140trka antibody or antiphosphotyrosine
antibody (kindly provided by Dr. Oreste Segatto, Rome, Italy).
Transfection of pCEP4-p75LNGFR in LNCaP Cells
LNCaP cells were seeded in 100-mm dishes or 24-well multiwell
plates (for cell counting studies) and transiently transfected with the
pCEP4 plasmid (kindly provided by Prof. G. Della Valle, University of
Bologna, Italy) containing the human p75LNGFR
cDNA. Cells were transfected using a liposome mixture complexed with
DNA in a ratio 12:1, as described (49 ). Control cultures were
transfected with the same vector lacking the DNA coding sequence for
p75LNGFR. Briefly, LNCaP cells, grown at
6570% confluency, were deprived of serum and incubated for 4 h
at 37 C with 7.2 µg/ml DNA complexed with liposomes. FCS was then
added back to the cultures and cells were maintained for 72 h in
the absence or presence of NGF, as specifically indicated, before
harvesting for cell counting, assessment of
p75LNGFR expression, or detection of apoptotic
death.
Assessment of the Expression of High- and Low-Affinity NGF
Receptors in LNCaP Cells
To control for p140trka expression, LNCaP
cells were harvested from the dish with a rubber policeman and fixed in
70% ethanol at 4 C, overnight. In parallel,
p75LNGFR-transfected cells to be checked for
p75LNGFR expression were detached from the dish
and used immediately. This protocol was chosen because of the known
requirement of an intact membrane for the
p75LNGFR. Cells were repeatedly washed and
incubated for 1 h at room temperature with either a mouse anti-p75
monoclonal antibody (10 µg/ml) or a rabbit
anti-p140trka polyclonal antibody (1 µg/ml).
This step was followed by incubation with fluorescein isothiocyanate
(FITC)-conjugated goat either antimouse (for
p75LNGFR detection) or antirabbit (for
p140trka) IgG, for 45 min at room temperature.
Controls included omission of the primary antibody and substitution
with nonimmune serum. Samples were analyzed using an Elite flow
cytometer (Coulter Electronics). At least 10,000 forward and side
scatter gated events were collected per specimen. Cells were excited at
488 nm, and the fluorescence was monitored at 525 nm. FITC fluorescence
was collected using logarithmic amplification.
Evaluation of Apoptotic Death in
p75LNGFR-Transfected LNCaP Cells
Externalization of phosphatidylserine on the cell membrane,
detectable by interaction with annexin V, which represents an early
index of apoptotic death, was evaluated by incubating harvested cells
with FITC-conjugated annexin V, at the concentration of 1 µg/ml for
15 min, according to manufacturers instructions (CLONTECH Laboratories, Inc., Palo Alto, CA). Immunofluorescence for
annexin V-FITC was analyzed with an Elite flow cytometer. At least
5,000 forward scatter gated events per specimen were collected. Annexin
V-FITC fluorescence was collected using logarithmic amplification.
The appearance of a prediploid cell population, indicative of damaged
and fragmented DNA, was evaluated cytofluorimetrically in LNCaP cells
stained with the nuclear dye propidium iodide. After fixation with 70%
ethanol, overnight at -20 C, cells were incubated with RNAse (100
µg/ml) for 2 h at 37 C and stained with propidium iodide (final
concentration, 50 µg/ml). Analysis was carried out in a flow
cytometer and restricted to cells with diploid and hypodiploid DNA
content.
Statistical Analysis
Data were analyzed by Students t test and, where
appropriate, by one- and two-way ANOVA as specifically indicated.
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to thank Alessandra Caruso and Salvatore Arena
for valuable advice and assistance during the establishment of
transfection conditions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Maria Angela Sortino, MD, Institute of Pharmacology, University of Catania School of Medicine, Viale Andrea Doria 6, 95125 Catania, Italy.
This work was supported in part by FAR (Fondi Ateneo Ricerca)
grants from the Universities of Catania (to M.A.S.) and Pavia (to
P.L.C.).
Received for publication December 16, 1998.
Revision received September 10, 1999.
Accepted for publication September 15, 1999.
 |
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