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The Prostate Centre, Vancouver General Hospital, Vancouver, British Columbia V6H 3Z6; and Division of Urology, University of British Columbia, Vancouver, British Columbia V5Z 3J5, Canada
Address all correspondence and requests for reprints to: Martin E. Gleave, Division of Urology, University of British Columbia, D-9, 2733 Heather Street, Vancouver, British Columbia V5Z 3J5, Canada. E-mail: gleave{at}unixg.ubc.ca
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Insulin-like growth factor (IGF)-I and IGF-II are potent mitogenic and antiapoptotic factors for various types of normal and malignant tissues. The biological response of cells to IGFs is regulated by several factors in the microenvironment, including the IGF binding proteins (IGFBPs). To date, at least six IGFBPs have been identified that modulate the biological action of the IGFs through high-affinity binding interactions that influence the ability of IGFs to function as ligands for the type-I IGF receptor (9, 10). However, IGFBP physiology is complex, as demonstrated by the findings that both stimulatory and inhibitory effects of IGFBPs on cell proliferation have been reported (9, 10, 11, 12, 13, 14) and that certain regulatory actions of IGFBPs are independent of IGFs (9, 10, 11, 15).
Accumulating evidence suggest that IGFBPs play an important role in the pathophysiology of prostate cells. Several IGFBPs are produced by normal prostate epithelial cells and/or stromal cells with rapid and dramatic alteration in the expression of certain IGFBPs after castration or treatment with antiandrogens (16, 17, 18, 19, 20). Changes in expression of various IGFBPs have also been reported in prostate cancer, with an increase in IGFBP-2 and IGFBP-5, and decrease in IGFBP-3 from the benign to malignant state (21, 22, 23). Furthermore, IGFBP-3 and IGFBP-4 have been shown to have apoptosis-inducing effects on prostate cancer cells (24, 25). However, despite undergoing the most substantial changes in expression in both benign and malignant prostate calls after androgen ablation, the functional significance of IGFBP-5 expression in prostate cancer has not been well characterized.
Controlled study of the complex molecular mechanisms associated with AI progression in prostate cancer has proved difficult because it cannot be replicated in vitro, and few animal models exist that reproducibly mimic the clinical course of the disease in men. Of the currently available human prostate cancer cell line, only LNCaP cells are androgen-responsive, prostate-specific antigen (PSA)-secreting, and immortalized in vitro. As in human prostate cancer, serum PSA levels in the LNCaP tumor model are initially regulated by androgen and directly proportional to tumor volume, and loss of maintenance of androgen-regulated PSA gene expression could be an endpoint of progression to androgen independence. Apoptotic tumor regression is not induced by castration; but tumor growth is inhibited, and serum PSA levels decrease by 80%, for several weeks after castration. After a prolonged period of growth in castrate hosts, LNCaP tumor growth rates increase, and PSA expression rises above precastrate level (26, 27). The LNCaP tumor model is, therefore, particularly useful in studying mechanisms controlling AI progression.
In this study, to clarify the functional significance of IGFBP-5 up-regulation after castration, IGFBP-5-overexpressing LNCaP cells were generated by stable transfection. We then evaluated the effects of IGFBP-5 overexpression on IGF-I-mediated mitogenesis, signal transduction, and time to progression to androgen-independence in the LNCaP tumor model.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression plasmid and transfection into LNCaP cells
Total RNA of human prostate cancer PC3 cells, which express
IGFBP-5 messenger RNA (mRNA) (29), was isolated by the
acid-guanidium thiocynate-phenol-chloroform method. The human
IGFBP-5 complementary DNA (cDNA) was generated by RT-PCR
from total RNA of PC3 cells by using the sense primer (5'-TAAAG
AAGCTTGACTAAGAGAAGATGGTGTT-3') containing a HIND III site
(underlined), and the antisense primer
(5'-GGTTGTCTAGAGACGCATCACT-CAACGTTGCT-3') containing a
XbaI site (underlined). PCR was performed with a
Cetus Gene Amp PCR system 9600 (Perkin-Elmer Corp.,
Norwalk, CT) in a 25-µl reaction vol, for 35 cycles, using rTth DNA
polymerase (Perkin-Elmer Corp.). Each cycle consisted of
denaturation at 95 C for 1 min, annealing at 60 C for 1 min, and
extension at 72 C for 1 min. The PCR fragments were double-digested
with HindIII and XbaI and then ligated into a
pRc/CMV expression vector (Invitrogen, Carlsbad, CA). The
correct sequence of a cloned fragment was confirmed by DNA
sequencing.
The pRc/CMV/IGFBP-5 construction was transfected into LNCaP cells by the liposome-mediated gene transfer method (30). Briefly, 2 x 105 LNCaP cells were plated in a 6-cm dish, 1 day before transfection. Five micrograms of purified pRc/CMV/IGFBP-5 or pRc/CMV (as a control) was added to LNCaP cells after a preincubation for 30 min with 5 µg of lipofectamine reagent and 3 ml of serum-free OPTI-MEM (Life Technologies, Inc.). Drug selection, in 300 µg/ml Genetisin (Life Technologies, Inc.), was begun 3 days after transfection. Three weeks after the drug selection, colonies were harvested with cloning cylinders and expanded to cell lines.
Northern blot analysis
Total RNA was isolated from cultured LNCaP sublines by the
acid-guanidium thiocynate-phenol-chloroform method. The
electrophoresis, hybridization, and washing conditions were carried out
as previously reported (31). Human IGFBP-5, cyclin D1, and
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA probes were
generated by RT-PCR from total RNA of PC3 cells using primers
5'-TGCGACGAGAAAGCCCTCTCCAT-3' (sense) and 5'-AAGGTTTGCACTGCTTTCTCTT-3'
(antisense) for IGFBP-5, 5'-TCCTACTTC-AAATGTGTGCAGAA-3' (sense) and
5'-TCACACTTGATCACTCTGGAGA-3' for cyclin D1, and
5'-TGCTTTTAACTCTGGTAAAGT-3' (sense) and 5'-ATATTTGGCAGGTTTTTCTAGA-3'
(antisense) for GAPDH. Density of bands for IGFBP-5 or cyclin D1 was
normalized against that of GAPDH by densitometric analysis.
Western blot analysis
Western analysis was performed as described previously (31).
Briefly, samples containing equal amounts of protein (25 µg) from
lysates of the cultured LNCaP sublines were electrophoresed on an
SDS-polyacrylamide gel and transferred to a nitrocellulose filter. The
filters were blocked in PBS containing 5% nonfat milk powder at 4 C
overnight and then incubated for 1 h with antibodies against
IGFBP-5 (Research Reagents, Webster, TX), ß-tubulin (Chemicon
International Inc., Tumecula, CA), and total and phospho-specific Akt
and mitogen-activated kinase (MAPK) (New England Biolabs, Inc., Boston, MA). The filters were then incubated for 30 min
with horseradish peroxidase-conjugated secondary antibodies
(Amersham Pharmacia Biotech, Arlington Heights, IL), and
specific proteins were detected using an enhanced chemiluminescence
Western blotting analysis system (Amersham Pharmacia Biotech).
In vitro mitogenic assay
In vitro mitogenic assay was performed as described
previously (27). Briefly, 3 x 103 cells
were seeded in each well of 96-well microtiter plates and allowed to
attach overnight. After exchanging the normal media for CSM with and
without 1 nM dihydrotestosterone (DHT)
(Sigma, St. Louis, MO), the LNCaP cell sublines were
treated with 10 ng/ml recombinant IGF-I (Sigma) or 10
µg/ml anti-IGF-I antibody (Upstate Biotechnology, Inc.,
Lake Placid, NY). After a 48-h incubation period, cells were fixed with
1% glutaraldehyde (Sigma Chemical Co.) and stained with
0.5% crystal violet (Sigma). The optical density was
determined with a microculture plate reader (Becton Dickinson and Co. Labware, Lincoln Park, NJ) at 540 nm. Absorbance values were
normalized to the values obtained for the vehicle-treated cells to
determine the percent of survival. Each assay was performed in
triplicate.
Flow cytometric analysis
The flow cytometric analysis of propidium iodide-stained nuclei
was performed as described previously (30). Briefly, the LNCaP cell
sublines were plated at densities of 5 x
106 cells in 6-cm dishes and treated as described
above. The cells were trypsinized 48 h after addition of
recombinant IGF-I or anti-IGF-I antibody, washed twice with PBS, and
fixed in 70% ethanol for 5 h at 4 C. The fixed cells were washed
twice with PBS, incubated with 1 µg/ml RNaseA (Sigma)
for 1 h at 37 C, and stained with 5 µg/ml propidium iodide
(Sigma) for 1 h at room temperature. The stained
cells were analyzed for relative DNA content on a FACScan (Becton Dickinson and Co. Labware).
DNA fragmentation assay
The nucleosomal DNA degradation was analyzed as described
previously, with a minor modification (30). Briefly, 1 x
105 of the LNCaP subline were seeded in 6-cm
culture dishes and, 24 h later, the medium was replaced with
serum-free conditioned medium containing either 50 µM
phosphatidylinositol 3'-kinase (PI3K) inhibitor, LY294002
(Sigma), or MAPK inhibitor, PD98059 (New England Biolabs, Inc.). After incubation for 1 h, the LNCaP
sublines were treated with 10 or 100 ng/ml recombinant IGF-I, 10 ng/ml
recombinant epidermal growth factor (EGF), 10 ng/ml recombinant basic
fibroblast growth factor, or 10 ng/ml recombinant keratinocyte growth
factor (KGF) for 24 h. The cells were then harvested and lysed in
a solution containing 100 mM NaCl, 10 mM Tris
(pH 7.4), 25 mM EDTA, and 0.5% SDS. After the
centrifugation, the supernatants were incubated with 300 µg/ml
proteinase K for 5 h at 65 C and extracted with phenol-chloroform.
The aqueous layer was treated with 0.1 vol of 3 M sodium
acetate, and the DNA was precipitated with 2.5 vol of 95% ethanol.
After treatment with 100 µg/ml RNaseA for 1 h at 37 C, the
sample was electrophoresed on a 2% agarose gel and stained with
ethidium bromide.
Assessment of in vivo tumor growth and determination of serum PSA
levels
One million cells of each LNCaP sublines were inoculated sc with
0.1 ml of Matrigel (Becton Dickinson and Co. Labware) in
the flank region of 6- to 8-week-old male athymic nude mice (BALB/c
strain; Charles River Laboratories, Inc., Montréal,
Québec, Canada). Each experimental group consisted of 6 mice.
Mice were castrated, via a scrotal approach, when tumors reached 100
and 200 mm3 in vol. Tumor volume was measured
once weekly and calculated as described previously (26). Mice were
maintained in accordance with institutional accredited guidelines of
the University of British Columbia.
Blood samples were obtained with tail vein incisions of mice once weekly. Serum PSA levels were determined by an enzymatic immunoassay kit with a lower limit of sensitivity of 0.2 µg/liter (Abbott Laboratories Canada Ltd., Montréal, Québec, Canada), according to the manufacture’s protocol. Data points were reported as mean values ± SD.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IGFBP-5
mRNA and protein were detected in four independent IGFBP-5-transfected
LNCaP cell lines (LNCaP/BP5a to LNCaP/BP5d). No detectable IGFBP-5
mRNA and protein were expressed in either the parental LNCaP (LNCaP/P)
or the control vector-transfected cell line (LNCaP/C). Because
growth rates were similar in all four clones expressing IGFBP-5, we
hereafter report only the data of LNCaP/P, LNCaP/C, LNCaP/BP5a, and
LNCaP/BP5b in subsequent experiments.
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, LNCaP/BP5a and LNCaP/BP5b cell
growth rates were significantly faster than those of LNCaP/P and
LNCaP/C cells, under both conditions, with and without DHT.
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,
the response of the various LNCaP sublines to IGF-I and anti-IGF-I
antibody treatment was dependent on the presence of DHT. In CSM without
DHT, the growth rates of all the LNCaP sublines were not affected by
recombinant IGF-I treatment. On the other hand, anti-IGF-I antibody
inhibited the growth of LNCaP/BP5a and LNCaP/BP5b cells but not that of
LNCaP/P and LNCaP/C cells. In contrast, in CSM with DHT, recombinant
IGF-I significantly increased the growth of LNCaP/P and LNCaP/C cells
but not that of LNCaP/BP5a and LNCaP/BP5b cells, and the growth of the
all LNCaP sublines was suppressed by anti-IGF-I treatment.
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, during the exponential phase of cell
growth, the growth rates of the LNCaP sublines, after treatment with
recombinant IGF-I or anti-IGF-I antibody, correlated directly with the
fraction of cells in the S + G2/M phases in both types of media.
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, cyclin D1 mRNA expression levels in
LNCaP/BP5a and LNCaP/BP5b cells were significantly higher than those in
LNCaP/P and LNCaP/C cells, in the presence and absence of 1
nM DHT. In addition, after treatment with recombinant IGF-I
and anti-IGF-I antibody, changes in cyclin D1 mRNA levels of the LNCaP
sublines paralleled changes in their growth rates and fraction in the S
+ G2/M phases in both types of media.
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, A and B).
In contrast, PI3K assays, using Akt as the downstream substrate, showed
that LNCaP/BP5a and LNCaP/BP5b cells had significantly higher PI3K
activity than LNCaP/P and LNCaP/C cells, in the presence of 1
nM DHT, and that changes in PI3K activity in the LNCaP
sublines, induced by recombinant IGF-I or anti-IGF-I antibody,
reflected changes in growth rates under conditions both with and
without 1 nM DHT (Fig. 4, C and D).
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). To assess
whether the prorective role of IGF-I against LY294002-induced apoptosis
in LNCaP/BP5a and LNCaP/BP5b cells was growth-factor-specific, the
effects of treatment with EGF, bFGF, and KGF on LY294002-induced
apoptosis in the LNCaP sublines were examined. EGF inhibited apoptosis
induced by LY294002 in all the LNCaP sublines, regardless of IGFBP-5
expression, whereas basic fibroblast growth factor and KGF had no
effect on LY294002-induced apoptosis in all the LNCaP sublines (data
not shown).
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). Serum PSA, in
mice bearing LNCaP/P and LNCaP/C tumors, decreased by 82% and
77%, respectively, by 2 weeks after castration and increased from 4–9
weeks after castration by 2.0- and 2.1-fold, respectively. In
comparison, serum PSA, in mice bearing LNCaP/BP5a and LNCaP/BP5b
tumors, decreased by 61% and 58%, respectively, before increasing
(beginning 2 weeks after castration) to 4.7- and 5.3-fold,
respectively, by 9 weeks. (Fig. 6B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, we reported dramatic increases in IGFBP-5 mRNA expression after castration in the androgen-dependent mouse Shionogi tumor model, and that IGFBP-5 mRNA expression is directly regulated by apoptosis-inducing stimuli rather than androgen withdrawal (40). Our results agree with previous reports that IGFBP-5 expression changes most substantially among the IGFBP family in prostate tissues after androgen ablation (16, 17, 18, 19, 20). Although various functional roles of IGFBP-5 expression have been reported in different model systems, these data are varying and conflicting. For example, IGFBP-5 has been reported to either stimulate or inhibit cell proliferation under different experimental conditions (10, 12, 13, 41, 42, 43, 44), and these effects occur dependent and/or independent of its well-characterized actions associated with modulation of IGF bioavailability (10, 12). Furthermore, to date, there has been no data demonstrating the functional significance of IGFBP-5 up-regulation after androgen ablation in prostate cancer progression.
In this study, we generated several IGFBP-5-overexpressing LNCaP cell lines to characterize the functional role of IGFBP-5 up-regulation in AI progression of prostate cancer. We found that IGFBP-5 overexpression confers a growth advantage upon LNCaP cells in both the presence and absence of DHT. Although IGFBP-5-negative control clones did not proliferate in the absence of DHT, the growth rates of IGFBP-5-transfected clones without DHT were similar to those of control clones with DHT, which suggests that IGFBP-5 overexpression partially rescues LNCaP cells from the growth arrest induced by androgen deprivation. We subsequently showed that the growth rates of IGFBP-5-negative control cells were increased by exogenous IGF-I and reduced by anti-IGF-I antibody in the presence of DHT, whereas these rates were not affected by either agent in the absence of DHT. In addition, the growth of IGFBP-5 transfectants was inhibited by anti-IGF-I antibody, but not stimulated by exogenous IGF-I, in both types of medium. Collectively, these findings suggest that IGFBP-5 overexpression reduces the minimal concentration requirement for IGF-I-mediated LNCaP cell growth. Although neutralization of IGF-I activity by anti-IGF-I antibody resulted in the substantial growth inhibition of both control and IGFBP-5-overexpressing LNCaP cells, the proliferation rate of IGFBP-5 transfectants remained higher than that of control cells, even in the absence of IGF-I activity, suggesting that IGFBP-5-regulated changes in LNCaP proliferation occurs via both IGF-I-dependent and -independent mechanisms.
Analysis of the two major signal transduction pathways (i.e. MAPK and PI3K pathways) for IGF-I (33, 34) revealed significant changes in PI3K activity, but not MAPK activity, in the LNCaP sublines. Changes in PI3K activity in the IGFBP-5 sublines, after treatment with recombinant IGF-I or anti-IGF-I antibody, reflected changes in their growth rates. Furthermore, treatment with the PI3K inhibitor, LY294002 (but not the MAPK inhibitor, PD98059) induced apoptotic cell death in the LNCaP sublines. Interestingly, LY294002-induced apoptosis in IGFBP-5 transfectants could be inhibited by IGF-I treatment, suggesting that IGFBP-5 can potentiate the antiapoptotic effects of IGF-I. Kulik et al. (34) reported that, when Rat-1 fibroblasts were pretreated with the PI3K inhibitor, wortmannin, IGF-I failed to protect them from apoptosis; however, when IGF-I receptors were overexpressed, IGF-I-mediated survival became largely insensitive to wortmannin. Although exogenous IGF-I increased PI3K activity (i.e. Akt phosphorylation) in IGFBP-5 transfectants in the absence of DHT, exogenous IGF-I did not increase PI3K above peak levels in the presence of DHT. We speculated that IGF-I-mediated PI3K signaling became more important when LNCaP cells were grown in the absence of androgen. Alternatively, PI3K activity in IGFBP-5 transfectants may be maximally stimulated when DHT is present, and addition of exogenous IGF-I has no further effect. Collectively, these findings suggest that IGF-mediated cell survival signaling in LNCaP cells occurs principally via the PI3K pathway, and that IGFBP-5 overexpression helps enhance IGF-I signaling in a similar fashion to the IGF-I receptor.
To test whether IGFBP-5 overexpression helps accelerate progression to androgen-independence, the LNCaP sublines were inoculated into male nude mice; and the changes in tumor volume and serum PSA levels were monitored before and after castration. Although tumor incidence, tumor growth rates, and serum PSA levels were similar among the LNCaP sublines growing in intact mice, tumor growth and serum PSA increased several fold faster in mice bearing IGFBP-5-transfected LNCaP tumors, after castration, than those bearing control LNCaP tumors. These results provide the first clear evidence that IGFBP-5 overexpression can increase tumor cell proliferation and accelerate time to AI progression after castration.
As outlined above, the biological activity of IGFBP-5 varies, depending upon various cell types, which may reflect differential regulation of extracellular matrix interactions (9, 19, 42, 43) or posttranslational modification (44). Indeed, in the Shionogi tumor model, IGFBP-5 expression is directly regulated by apoptotic stimuli and dramatically up-regulated by castration (40); whereas, in the CWR22 tumor model, castration decreases IGFBP-5 expression, and the changes in IGFBP-5 expression parallel changes in androgen levels (20). Although studies using additional prostate tumor systems are needed to clarify tissue-specific interactions between IGF-I and IGFBP-5, and to better define the relative importance of IGFBP-5 after androgen ablation in prostate cancer, the present study provides the first functional evidence that overexpression of IGFBP-5 potentiates the mitogenic and antiapoptotic activities of IGF-I through enhanced PI3K pathway, thereby serving as one mechanism capable of accelerating progression to androgen independence.
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Acknowledgments |
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Footnotes |
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Received November 11, 1999.
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