| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St Leonards, New South Wales 2065, Australia
Address all correspondence and requests for reprints to: Dr. Janet Martin, Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, University of Sydney, New South Wales, 2065 Australia. E-mail: janetlm{at}med.usyd.edu.au
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The IGFBP family comprises six structurally related proteins,
designated IGFBP-1 to -6, having high affinity
(
1010-1011 liter/mol)
for both IGF-I and IGF-II (5). Various studies have demonstrated the
production of one or more IGFBPs by normal and malignant prostate
epithelial cells (3, 6, 7, 8), and three cell lines frequently used as
models of prostate carcinoma, PC-3, Du145, and LNCaP, also secrete
IGFBPs (7, 9, 10, 11, 12, 13, 14). Modulation of IGF bioactivity by endogenous or
exogenous IGFBPs has been demonstrated in the PC-3 and Du145 cell lines
(12, 13, 15), and recent evidence indicates that some of the IGFBPs may
also exert growth-inhibitory effects independently of preventing type 1
IGF receptor activation. In the PC-3 cell line, IGFBP-3 is reported to
both inhibit cell proliferation (8) and induce apoptosis (16) via
IGF-receptor-independent pathways. These findings point to a
significant role for IGFBP-3 in the regulation of prostate cancer cell
proliferation.
In the rat prostate in vivo and prostate carcinoma cells in vitro, 1,25-dihydroxyvitamin D3 (vitamin D) and its analog EB1089 decrease cell proliferation concomitantly with increased IGFBP-3 expression (15, 17), suggesting that in prostate cancer cells, as in other cell systems (18), IGFBP-3 may mediate the growth-inhibitory effect of vitamin D. Other studies have shown an interaction between vitamin D and dihydrotestosterone (DHT) in the modulation of prostate cell proliferation (19), but the role of IGFBP-3 in this is not clear. In the present study we have used the androgen-responsive LNCaP prostate carcinoma cell line to investigate the regulation of IGFBP-3 production by these agents, and the role of endogenous IGFBP-3 in LNCaP cell proliferation. Our data show that unexpectedly, endogenous and exogenous IGFBP-3 can stimulate DNA synthesis and cell proliferation in the LNCaP cell line.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The IgG fraction from IGFBP-3 polyclonal antiserum was isolated by protein A-affinity chromatography. One milliliter serum (R30) was adsorbed onto a column containing 2 ml protein A Sepharose (Amrad Pharmacia Biotech). After 1 h at 22 C, the column was washed with 50 ml of 50 mM sodium phosphate (pH 6.5) containing 0.1 M NaCl, and eluted with 10 ml 0.1 M glycine, pH 3. The eluted IgG fraction was adjusted to starting volume (1 ml) by centrifugation through a Centricon 30 ultrafiltration device (Amicon, Inc., Beverly, MA) and equilibrated to pH 7.4 with RPMI medium. Nonimmune rabbit serum for use as a control was prepared in the same way.
Cell cultures
LNCaP cells were purchased from the American Type Culture Collection (Manassas, VA), maintained in RPMI medium containing
5% FCS, 2 mM glutamine, and 10 µg/ml bovine insulin, and
passaged by trypsinization every 6–7 days. For stimulation
experiments, confluent cell monolayers in 24-well plates were incubated
for 48 h in serum- and insulin-free RPMI medium containing 2
mM glutamine and 1 g/liter BSA [serum-free medium
(SFM)] before addition of test reagents in fresh SFM for 1–4 days.
Media were collected for analysis of IGFBPs by RIA and Western
blotting, and cell monolayers were processed for cell-associated IGFBPs
as described below.
RIAs and IGFBP-3 cell association assay
IGFBP-2 (22) and IGFBP-3 (21) in cell-conditioned medium were
measured by RIA as previously described. Cell- or matrix-associated
IGFBP-3 was measured immunologically as described previously (23).
Briefly, cell monolayers were rinsed in SFM, and then incubated for
16 h at 22 C with anti-IGFBP-3 serum at a final dilution of
1:5,000 in SFM. Monolayers were washed twice with SFM and incubated for
an additional 2 h with [125I]protein A
(20,000 cpm/well in SFM). Monolayers were again washed, solubilized in
5 g/liter SDS, and then lysates were 
-counted. Nonspecific binding
was determined using normal rabbit serum in lieu of immune serum. This
assay cannot distinguish between IGFBP-3 bound with the cell and that
associated with the extracellular matrix.
Northern analysis
LNCaP RNA was isolated using Total RNA Isolation Reagent
(Advanced Biotechnologies, Surrey, UK) and quantitated by absorbance at
260 nm. Northern analysis was carried out as previously described (23)
using the [32P]dCTP-labeled complementary DNA
probe for human IGFBP-3 (a gift of Dr. N. Shimasaki, University of
California San Diego, La Jolla, CA). Filters were hybridized overnight
at 42 C, and then washed in 0.1x SSC at 42 C, with an additional wash
in 1x SSC if required. Filters were quantified using a PhosphorImager
(Molecular Dynamics, Inc., Sunnyvale, CA).
SDS-PAGE and Western blotting
Electrophoresis and Western blotting were carried out as
previously described (24). Conditioned media were prepared for Western
blot analysis of IGFBPs by 5-fold concentration through Centricon
10 ultrafiltration units (Amicon Inc., Beverley, MA).
Concentrates (50 µl) were mixed with 10 µl sample buffer and
electrophoresed through 12% gels at 80 V for 16 h. Separated
proteins were transferred to Hybond C membrane, and then membranes were
blocked in Tris-buffered saline (TBS, 10 mM Tris, 50
mM sodium chloride, pH 7.4) containing 10 g/liter BSA, 0.2
g/liter sodium azide, and 0.5 ml/liter Nonidet P-40 ("blocking
buffer") for 2–3 h at 22 C. For immunoblotting, blocked membranes
were incubated overnight at 4 C with IGFBP-3 antiserum (R30) at 1:5,000
final dilution, or IGFBP-2 antiserum (22) at 1:2,000 final dilution.
Membranes were then rinsed briefly in TBS containing 0.05% Nonidet
P-40 and incubated for a further 2 h with
[125I]protein A (1 x
106 cpm/50 ml in blocking buffer). Blots were
washed three times for 10 min each in TBS containing Nonidet P-40, air
dried, and autoradiographed for 4 days at -70 C.
Thymidine incorporation and cell proliferation assays
DNA synthesis was assessed by incorporation of
[3H]thymidine. Cell monolayers in 24-well
plates were serum-starved for 48 h before the start of the
experiment. Treatments (vitamin D, DHT, antibodies, etc.) were added
for 24 h in 0.5 ml SFM. During the final 4 h of this period,
1 µCi/well [3H]thymidine (35 Ci/mmol, ICN
Biochemicals, Inc., Cleveland, OH) was added in 50 µl SFM.
Monolayers were rinsed twice with ice-cold saline and fixed with 1
ml/well ice-cold methanol-acetic acid (3:1) at 4 C for a minimum of
2 h. Cells were solubilized in 0.5 ml of 5 g/liter SDS, and 250
µl of each lysate were mixed with scintillant (OptimaGold,
Hewlett-Packard Co., Palo Alto, CA) before counting for 2
min. Cell proliferation was determined after treatment for 4 days.
Confluent monolayers in six-well multidishes were treated as described
above with additions made in 4 ml medium/well. Four days later, media
were removed, and cells were dispersed using trypsin-EDTA. Aliquots of
suspended cells were counted using a hemocytometer.
Statistical Analysis
Individual experiments were conducted in triplicate or
quadruplicate wells; experiments were carried out at least three times
unless indicated otherwise. Data were analyzed by ANOVA and Fisher’s
protected least significant difference test using the StatView program
for Macintosh (SAS Institute, Inc., Cary, NC); differences
were considered significant where P < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). The more
abundant of these proteins, with a molecular mass of approximately 34
kDa, was determined by immunoblotting to be IGFBP-2, while the
broad band of 43–45 kDa was IGFBP-3. RIA of these proteins in
unconcentrated media confirmed that IGFBP-2 levels were approximately
25- to 30-fold higher than IGFBP-3. Determined in six experiments,
confluent monolayers secreted 16.7 ± 1.03 ng/ml (mean ±
SE) IGFBP-2, and 0.67 ± 0.04 ng/ml IGFBP-3 over
24 h under serum-free conditions.
|
, 24 h-treatment with DHT increased
medium concentrations of both IGFBP-2 (panel 2A) and IGFBP-3 (panel 2B)
by approximately 50% (P < 0.01 compared with control
for both). A significant increase in IGFBP-3 was also observed in
response to treatment with 10 nM vitamin D,
resulting in levels 2-fold elevated compared with untreated cells
(P < 0.01). A synergistic effect of the two agents was
indicated by markedly elevated IGFBP-3 levels, with a combination of
DHT and vitamin D resulting in a 4- to 5-fold increase in medium
IGFBP-3 concentrations. By contrast, vitamin D alone had no effect on
IGFBP-2, nor did it alter the response to DHT (P = 0.52
for DHT treatment vs. DHT plus vitamin D). When IGFBP-3
concentrations were determined 4 days after addition of agents, DHT was
no longer stimulatory for IGFBP-3 production in the absence of
vitamin D and did not enhance the stimulatory effect of vitamin D on
IGFBP-3 production (Fig. 2C). However, if agents were added daily for
the 4-day incubation period, DHT once again stimulated IGFBP-3 levels
in the absence and presence of vitamin D (Fig. 2D).
|
). In addition, the presence of
smaller immunoreactive species, of 30 kDa and 20 kDa, was apparent in
media conditioned by cells treated with a combination of DHT and
vitamin D. These fragments were not detectable by IGF-I or IGF-II
ligand blotting (not shown).
|
) indicated that in the absence of
vitamin D, a significant increase (P < 0.05) in
secreted IGFBP-3 was apparent with 1 nM DHT, with
levels elevated more than 2-fold at 100 nM DHT.
Inclusion of 10 nM vitamin D markedly potentiated
the stimulatory effects of DHT, increasing IGFBP-3 more than 7-fold at
100 nM DHT (P < 0.001 compared
with control or 10 nM vitamin D). When increasing
doses of vitamin D were tested in the absence of DHT (Fig. 4B), a
significant increase in IGFBP-3 was apparent at 1
nM vitamin D and reached a maximum at 10
nM vitamin D. Coincubation with 10
nM DHT augmented the vitamin D effect, with
half-maximal stimulation of IGFBP-3 at approximately 1
nM vitamin D in the presence or absence of DHT.
Northern analysis of IGFBP-3 messenger RNA (mRNA) (Fig. 4C) indicated a
single hybridizing band of 2.5 kb, which was slightly increased by 10
and 100 nM DHT compared with control; vitamin D
(10 nM) also slightly increased the level of
IGFBP-3 mRNA (Fig. 4C, lane 4). A marked increase in IGFBP-3 mRNA
was apparent for the combined treatment of 10 nM
vitamin D and 100 nM DHT (Fig. 4C, lane 6),
suggesting that the 8-fold increase in IGFBP-3 immunoreactivity
detected in medium resulted from increased expression of IGFBP-3
mRNA.
|
, preincubation with vitamin D followed
by exposure to DHT resulted in markedly elevated IGFBP-3 concentrations
(9-fold compared with control, P < 0.0001), and this
was significantly higher than in media where DHT was added in both the
pretreatment and conditioning periods (P < 0.0001). By
contrast, the response to vitamin D was the same regardless of whether
DHT or vitamin D was included in the preincubation period
(P = 0.82). These results suggest that, in LNCaP cells,
vitamin D enhances DHT-stimulated IGFBP-3 secretion by increasing
responsiveness to DHT.
|
, cell-associated IGFBP-3 was not
significantly altered in response to 10 nM DHT
(P = 0.41); however, vitamin D (10
nM) caused a 25% increase in the amount of
IGFBP-3 associated with the cell or matrix (P <
0.001). This was further elevated, to 150% of control, in the presence
of 10 nM DHT (P < 0.001 for DHT
+ vitamin D vs. vitamin D alone). Addition of IGF-I (50
ng/ml) subsequent to incubation with DHT plus vitamin D resulted in a
reduction in the amount of cell- or matrix-associated IGFBP-3 (Fig. 6A). By contrast, [LR3]IGF-I, an IGF analog
that has reduced affinity for IGFBP-3, had no significant effect.
|
) with a significant effect
(P < 0.0001 compared with no addition) apparent at 100
ng/ml IGFBP-3. As before, DHT and vitamin D (10
nM each) significantly increased cell- or
matrix-associated IGFBP-3 in the absence of exogenous IGFBP-3 (Fig. 6B). In the presence of vitamin D and DHT, a significant increase in
the level of cell- or matrix-associated IGFBP-3 was apparent at a lower
concentration of exogenous IGFBP-3 (10 ng/ml) (Fig. 6B;
P < 0.05 compared with DHT plus vitamin D). However,
this effect was lost with increasing concentrations of IGFBP-3, so that
at 1000 ng/ml exogenous IGFBP-3, there was significantly less
cell-associated IGFBP-3 detectable in the presence of DHT and vitamin D
than in their absence (P < 0.0001).
In other cells, increased secretion of IGFBP-3 may result in decreased
sensitivity to the proliferative effects of IGFs due to their
sequestration (24, 27). To examine how endogenous IGFBP-3 affected the
actions of IGFs in LNCaP cells, we examined basal and IGF-stimulated
[3H]thymidine incorporation in the absence and
presence of DHT and vitamin D. As shown in Table 1, LNCaP cells were insensitive to IGF-I,
with 50 ng/ml failing to stimulate DNA synthesis; higher concentrations
were similarly without effect. This lack of response to IGF-I was not
due to high levels of endogenous IGFBPs because
[LR3]IGF-I, the IGF analog that does not bind
IGFBPs and should therefore be unaffected by their presence, similarly
failed to stimulate DNA synthesis (Table 1). When added separately at
10 nM, DHT and vitamin D caused a slight increase in DNA
synthesis; however, this was not statistically significant (data not
shown). The combination of DHT and vitamin D each at 10 nM
caused a 3-fold increase in DNA synthesis compared with control (Table 1, P < 0.0001). Although neither IGF-I nor
[LR3]IGF-I significantly affected the response
to vitamin D plus DHT (Table 1), a trend toward decreased DNA synthesis
was noted with IGF-I (P = 0.084 for DHT + vitamin D +
IGF-I compared with DHT + vitamin D), but not
[LR3]IGF-I (P = 0.69, Table 1).
|
).
Higher concentrations of antibody did not result in a further decrease
in DNA synthesis (data not shown).
|
, cell-derived IGFBP-2 was not depleted from conditioned medium
(either untreated or DHT + vitamin D-treated) by IGFBP-3 antiserum
(Fig. 7B, lanes 1–4); similarly, pure IGFBP-2 could not be
precipitated by this antibody (Fig. 7B, lanes 5 and 6). Under these
conditions, significant depletion of cell-derived IGFBP-3 from medium
of DHT + vitamin D-treated cells (Fig. 7B, lanes 7 and 8), or pure
IGFBP-3 spiked into unconditioned medium (lanes 9 and 10), was
apparent.
The effect of plasma-derived IGFBP-3 on basal and stimulated thymidine
incorporation was then determined. As shown in Fig. 8A, thymidine incorporation was
significantly increased in the presence of 50 ng/ml pure IGFBP-3,
resulting in levels approximately 2-fold elevated compared with control
(P < 0.001); no significant effect was observed with
lower concentrations of IGFBP-3. Cells treated with a combination of
DHT, vitamin D, and IGFBP-3 showed a significant increase in DNA
synthesis relative to that seen with DHT and vitamin D alone
(P < 0.01). Exogenous IGFBP-2 at the same
concentration was without effect (data not shown).
|
, the pattern of change in cell
number was identical with that of DNA synthesis, although the magnitude
of the increase was smaller. IGFBP-3 (50 ng/ml) increased cell number
by 36 ± 7% (mean ± SE, pooled data from 2
experiments carried out in triplicate: P < 0.01
compared with control), while the combination of DHT plus vitamin D
resulted in a 26 ± 5% increase (P < 0.05
vs. control). Cell numbers did not change when DHT and
vitamin D were added separately (data not shown). The combination of
DHT, vitamin D, and IGFBP-3 resulted in an increase to 179 ± 11%
of control (P < 0.0001). Taken together, these results
indicate that endogenous and exogenous IGFBP-3 stimulate DNA synthesis
and cell proliferation in LNCaP cells. Finally, we examined whether there was a relationship between the stimulatory effect of exogenous IGFBP-3 and its cell or matrix association. LNCaP cells were treated with 50 ng/ml IGFBP-3 in the presence of graded concentrations of IGF-I, after which DNA synthesis and cell-associated IGFBP-3 were analyzed. IGFBP-3 (50 ng/ml) increased DNA synthesis to 253 ± 62% of control (mean ± SE of pooled data from quadruplicate wells in two experiments). Coincubation with up to 100 ng/ml IGF-I reduced this to 182 ± 43% of control at the highest concentration of IGF-I tested; this was not statistically significant (P = 0.12). However, 100 ng/ml IGF-I fully blocked binding of 50 ng/ml IGFBP-3 to the monolayer, reducing cell- or matrix associated IGFBP-3 from 143 ± 6% control in the absence of IGF-I, to 112 ± 8% of control in its presence (P < 0.01).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Positive regulation of IGFBP-3 in response to DHT has also been shown in neonatal foreskin fibroblasts (31) and human immortalized osteoblastic cells (32). By contrast, DHT has been reported to decrease IGFBP-3 mRNA in LNCaP cells (33), and in PC-3 prostate carcinoma cells transfected with a constitutively active androgen receptor (34). A putative androgen response element has been identified in the DNA sequence of the human IGFBP-3 gene promoter between nucleotides -1742 and -1725 (35); to date, however, binding and activation of this sequence has not been reported. It may be significant that the studies showing reduced IGFBP-3 expression, either in response to DHT or in androgen receptor-activated cells, were carried out in the presence of charcoal-stripped FCS, while positive regulation of IGFBP-3 occurs in the absence of serum. This may indicate the involvement of serum-derived factors in the regulation of IGFBP-3 by androgens.
A marked effect on IGFBP-3 production was apparent when cells were treated either with a combination of vitamin D and DHT, or with DHT after preincubation with vitamin D. Our data indicated that vitamin D increased sensitivity to DHT, and are consistent with findings from other studies demonstrating that androgen receptors are up-regulated by vitamin D in LNCaP cells (25, 26). Zhao et al. (36) showed that androgens and vitamin D synergistically stimulate production of prostate-specific antigen (PSA) by LNCaP cells and that the effects of vitamin D on PSA production are androgen-dependent (36). Thus, it appears that although vitamin D is an independent regulator of IGFBP-3, in androgen-responsive cells it may also markedly increase IGFBP-3 production by enhancing the stimulatory effect of DHT.
The predominant form of immunoreactive IGFBP-3 detected in LNCaP-conditioned medium was the intact 43- to 45-kDa doublet; however, significant amounts of smaller immunoreactive, non-IGF-binding IGFBP-3 fragments of 30 and 20 kDa were present in medium from vitamin D plus DHT-treated cells. These fragments were detected in unacidified medium, indicating that they did not derive from the cathepsin D-mediated proteolysis of IGFBP-3 previously described in LNCaP cells (11). PSA, which is secreted by LNCaP cells, has also been shown to degrade IGFBP-3 (37), and its expression is increased in response to DHT and vitamin D (25, 36). However, the IGFBP-3 fragments of 20 and 30 kDa detected in the present study differ in size to those reported for proteolysis of CHO-derived IGFBP-3 by PSA—a major band of 25 kDa, with minor species of 35 and 30 kDa (37)—implying the activity of different enzyme(s). In the androgen receptor-negative prostate carcinoma cell line PC-3, a predominant IGFBP-3 fragment of 30 kDa generated by the action of serine proteases was detected in conditioned medium from untreated cells (12). We are now investigating whether the protease secreted by LNCaP cells is that enzyme.
Other studies have shown that LNCaP cells exhibit a biphasic response to androgens, with growth stimulation at low androgen concentration (1 nM), and growth inhibition at higher concentrations (38). A similar bell-shaped growth curve in response to vitamin D in the presence of DHT has also been shown in LNCaP cells (39). We also observed growth stimulation in response to the combination of vitamin D and DHT at concentrations (10 nM) previously reported to inhibit cell proliferation (26, 36, 38). While the cause of this discrepancy is not known, differential sensitivity to growth regulators may indicate the emergence of variant sublines of the parental LNCaP cells in different laboratories. Alternatively, it is possible that the experimental conditions used in the different studies may give rise to different results. Our studies were conducted in the absence of serum; however, the earlier studies of Lee et al. (38) and Zhao et al. (36) were carried out in the presence of charcoal-stripped FCS, which might contain factors that alter sensitivity to the growth-inhibitory effects of vitamin D and DHT.
Increased cell-associated IGFBP-3 in response to hormonal stimulation has not previously been described, although it is well recognized that IGFs can dissociate IGFBP-3 from the cell surface or extracellular matrix. In human fibroblasts that secrete high levels of IGFBP-3, the amount of cell-associated protein is not further increased by factors that increase extracellular IGFBP-3, such as transforming growth factor-ß1 (23). This may reflect saturation of binding sites for IGFBP-3 on the cell or matrix (40). By contrast, LNCaP cells secrete relatively little IGFBP-3 under serum-free conditions, and we found that in addition to increasing secreted IGFBP-3, the combination of DHT and vitamin D increased the amount of IGFBP-3 associated with the cell monolayer. This suggests that in these cells, an increase in IGFBP-3 expression may result in partitioning between secreted and cell-bound forms.
Dose-dependent binding of exogenous IGFBP-3 to LNCaP monolayers was also apparent in the absence and presence of DHT and vitamin D. When added at a low concentration (10 ng/ml), exogenous IGFBP-3 bound better in the presence of DHT and vitamin D than in their absence, suggesting that DHT and vitamin D facilitate IGFBP-3 binding to the cell surface or extracellular matrix. This effect was lost at high IGFBP-3 concentrations, however, implying that there are factors limiting the extent to which this enhancement of cell- or matrix association can occur. It is possible that hormones such as DHT and vitamin D modulate the binding sites with which IGFBP-3 interacts, or extracellular factors that regulate such interactions. Although IGFBP-3-binding species of varying size have been demonstrated in cell lysates or membrane preparations of some breast and prostate cancer cells (16, 41), their identity and signal transduction capability remain unknown.
An important finding of the present study was that the
cell-proliferative effects of DHT and vitamin D in LNCaP cells are
partly mediated by IGFBP-3. Immunoneutralization of endogenous IGFBP-3
in DHT and vitamin D-treated cells resulted in decreased DNA synthesis,
although it was not reduced to basal levels even at high concentrations
of antibody. In support of a growth-stimulatory role for IGFBP-3 in
these cells, plasma-derived IGFBP-3 also increased DNA synthesis and
cell number. It was interesting to note that the concentration of
exogenous IGFBP-3 required to elicit a response was significantly more
than the 3 ng/ml increase in extracellular IGFBP-3 brought about by
DHT and vitamin D. Whether this is due to a difference in bioactivity
between plasma- and cell-derived IGFBP-3 is currently under
investigation.
Alternatively, it is possible that the amount of IGFBP-3 associated with the cell or extracellular matrix, rather than the amount secreted, is an important determinant of its ability to stimulate DNA synthesis. In support of this, the magnitude of the change in cell/matrix-associated IGFBP-3 induced by DHT plus vitamin D was similar to that achieved by approximately 50 ng/ml exogenous IGFBP-3, and both treatments resulted in a similar level of increase in DNA synthesis. However, we also found that although IGF-I reduced the amount of cell- or matrix-associated IGFBP-3, this was not accompanied by a significant decrease in DNA synthesis, although a trend toward it was observed. This would suggest that the degree of stimulation of DNA synthesis by IGFBP-3 is not determined simply by how much IGFBP-3 is associated with the cell or extracellular matrix.
It has also been suggested that proteolysis of IGFBP-3 and subsequent release of IGFs from sequestration may be the mechanism involved in its growth-stimulatory effect (12, 42): IGFBP-3 potentiation of IGF-I action has been shown in fibroblasts (43, 44), MCF-7 breast cancer cells (45), and an immortalized osteoblast cell line (32). We found that IGFBP-3 proteolysis was increased in response to DHT plus vitamin D, but as LNCaP cells do not secrete IGFs (46), and a stimulatory effect of IGFBP-3 was seen in the absence of exogenous IGF-I or -II, an IGF-receptor-mediated mechanism of action appears unlikely. Furthermore, in our hands LNCaP cells were unresponsive to IGFs regardless of the presence of DHT and/or vitamin D, which is in contrast with the study of Iwamura et al. (46) who reported increased responsiveness to exogenous IGF-I in the presence of DHT. This discrepancy may provide further evidence of differences in the LNCaP cell cultures under investigation in different laboratories.
In summary, we have shown that vitamin D and DHT regulate the secretion, proteolysis, and cell- or matrix association of IGFBP-3 in LNCaP cells, and that both endogenous and exogenous IGFBP-3 can stimulate LNCaP DNA synthesis and cell proliferation. Defining the mechanism of action of IGFBP-3 in these cells and understanding the role of the different forms of IGFBP-3 in the regulation of normal and cancer cell growth are crucial areas for future investigation.
|
Footnotes |
|---|
Received August 26, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
,25-dihydroxycholecalciferol, all-trans
retinoic acid, 9-cis retinoic acid, and phenylacetate. Prostate 28:182–194[CrossRef][Medline]
,25-dihydroxyvitamin D3 in the
androgen-dependent LNCaP cells. Biochem Biophys Res Commun 223:141–146[CrossRef][Medline]
,25-dihydroxyvitamin
D3 and 9-cis retinoic acid in LNCaP human
prostate cancer cells. Endocrinology 140:1205–1212,25-Dihydroxyvitamin D3 actions in LNCaP
human prostate cancer cells are androgen-dependent. Endocrinology 138:3290–3298,25-dihydroxyvitamin D3. Cancer Res 52:515–520This article has been cited by other articles:
 |
|
 |
|
  J. L. Martin and R. C. Baxter Expression of Insulin-Like Growth Factor Binding Protein-2 by MCF-7 Breast Cancer Cells Is Regulated through the Phosphatidylinositol 3-Kinase/AKT/Mammalian Target of Rapamycin Pathway Endocrinology, May 1, 2007; 148(5): 2532 - 2541. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Seenundun and B. Robaire Time-Dependent Rescue of Gene Expression by Androgens in the Mouse Proximal Caput Epididymidis-1 Cell Line after Androgen Withdrawal Endocrinology, January 1, 2007; 148(1): 173 - 188. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Peng, P. J. Malloy, J. Wang, and D. Feldman Growth Inhibitory Concentrations of Androgens Up-Regulate Insulin-Like Growth Factor Binding Protein-3 Expression via an Androgen Response Element in LNCaP Human Prostate Cancer Cells Endocrinology, October 1, 2006; 147(10): 4599 - 4607. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Burrows, J. M. P. Holly, N. J. Laurence, E. G. Vernon, J. V. Carter, M. A. Clark, J. McIntosh, C. McCaig, Z. E. Winters, and C. M. Perks Insulin-Like Growth Factor Binding Protein 3 Has Opposing Actions on Malignant and Nonmalignant Breast Epithelial Cells that Are Each Reversible and Dependent upon Cholesterol-Stabilized Integrin Receptor Complexes Endocrinology, July 1, 2006; 147(7): 3484 - 3500. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. V. Desai, A. M. Michalowska, P. Kondaiah, J. M. Ward, J. H. Shih, and J. E. Green Gene Expression Profiling Identifies a Unique Androgen-Mediated Inflammatory/Immune Signature and a PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome 10)-Mediated Apoptotic Response Specific to the Rat Ventral Prostate Mol. Endocrinol., December 1, 2004; 18(12): 2895 - 2907. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. V. Stewart and N. L. Weigel Vitamin D and Prostate Cancer Experimental Biology and Medicine, April 1, 2004; 229(4): 277 - 284. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Yu, X.-O. Shu, B. D. L. Li, Q. Dai, Y.-T. Gao, F. Jin, and W. Zheng Joint Effect of Insulin-like Growth Factors and Sex Steroids on Breast Cancer Risk Cancer Epidemiol. Biomarkers Prev., October 1, 2003; 12(10): 1067 - 1073. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Martin, S. M. Weenink, and R. C. Baxter Insulin-like Growth Factor-binding Protein-3 Potentiates Epidermal Growth Factor Action in MCF-10A Mammary Epithelial Cells. INVOLVEMENT OF p44/42 AND p38 MITOGEN-ACTIVATED PROTEIN KINASES J. Biol. Chem., January 24, 2003; 278(5): 2969 - 2976. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Hoeflich, R. Reisinger, H. Lahm, W. Kiess, W. F. Blum, H. J. Kolb, M. M. Weber, and E. Wolf Insulin-like Growth Factor-binding Protein 2 in Tumorigenesis: Protector or Promoter? Cancer Res., December 1, 2001; 61(24): 8601 - 8610. [Full Text] [PDF]
|
|
|
|
 |
|
 G. A. BROCKMANN, C. S. HALEY, E. WOLF, S. KARLE, J. KRATZSCH, U. RENNE, M. SCHWERIN, and A. HOEFLICH Genome-wide search for loci controlling serum IGF binding protein levels of mice FASEB J, April 1, 2001; 15(6): 978 - 987. [Abstract] [Full Text] [PDF]
|
|
| ||||
