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The Laboratories for Reproductive Biology (C.W.G., F.S.F.), The Departments of Surgery (Division of Urology) (D.K., J.L.M.) and Pediatrics (C.W.G., F.S.F., P.Y., A.J.D.) and The Lineberger Comprehensive Cancer Center (A.J.D., J.L.M., F.S.F.), The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, Department of Pathology, Case Western Reserve University, Cleveland, Ohio (T.G.P.)
Address all correspondence and requests for reprints to: Frank S. French, The Laboratories for Reproductive Biology, Department of Pediatrics, CB 7500, 382 MSRB, Chapel Hill, North Carolina 27599. E-mail: fsfrench{at}med.unc.edu
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Introduction |
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Insulin-like growth factor (IGF)-I and its cognate receptor have been implicated in CaP cell growth (2, 3, 4). IGF-I is not produced at detectable levels by prostate epithelial cells, however prostate cells express the type I IGF receptor. Prostate epithelial cells make IGF-II but lack the type II IGF receptor (4). IGF receptor-mediated signaling appears essential to growth of CaP cells because cell proliferation can be inhibited by antibodies directed against IGF-I receptors (5), peptide analogues of IGF-I that block IGF-I receptor function (6), or a stably transfected antisense RNA expression vector that blocks IGF-I receptor expression (7). IGF-I receptor binding was demonstrated in human benign hyperplastic prostate (8). Recent studies by Chan et al. (9) demonstrated a positive correlation of serum IGF-I levels and risk of CaP. Men with high serum IGF-I levels (294–500 ng/ml) were 4.3 times more likely to develop CaP than were men with low serum levels of IGF-I. On the other hand, IGFBP-3 levels correlated inversely with the development of CaP suggesting lower serum IGFBP-3 allows greater bioavailability of IGF-I.
IGF binding proteins (IGFBPs) are secretory proteins that have autocrine and paracrine functions as modulators of IGF action. They have both inhibitory and potentiating effects on IGF actions (10). Moreover, recent studies suggest that some IGFBPs may have their own receptor-mediated functions independent of the IGF receptors (11). IGFBP-5 has been shown to potentiate IGF action (12).
In CaP, the majority of androgen-dependent and androgen-independent cells express AR protein. In clinically localized CaP treated with complete androgen blockade before radical prostatectomy, AR messenger RNA (mRNA) expression correlated directly with pathologic stage and Gleason grade (13). Hobisch et al. (14) found that all androgen-independent CaP (n = 22) examined immunohistochemically were AR positive. AR expression levels differ little between androgen-dependent and androgen-independent CaP (our unpublished results and 14, 15). In a subset of androgen-independent CaP, AR expression may be enhanced by gene amplification. AR gene amplification was reported in 30% of recurrent tumors (7 of 23), with no amplification detected before androgen deprivation therapy (16).
The human CaP xenograft, CWR22, is propagated sc in nude mice and retains biological characteristics exhibited by most human CaPs, including regression following androgen withdrawal and recurrence after several months in the absence of testicular androgen (17, 18, 19, 20). The recurrent tumor expresses a level of AR equal to or higher than that of the androgen withdrawn CWR22 and exhibits nuclear AR immunostaining. CWR22 contains a mutant AR (His874Tyr) with altered ligand specificity. The mutant AR exhibits a wild-type AR response to testosterone and dihydrotestosterone but is more responsive than wild-type AR to activation by estradiol and dehydroepiandrosterone (20). Using this human CaP xenograft, we examined the expression and androgen regulation of the IGFBPs. IGFBP-2, -3, -5, and -6 were expressed in both androgen-dependent and androgen-independent CWR22 but only IGFBP-5 was androgen-regulated. In prostate specimens from patients we found that immunostaining of IGFBP-5 was stronger in CaP than in BPH.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specimens of human benign or malignant prostate tissue obtained by transurethral resection or radical retropubic prostatectomy were fixed in 10% buffered formalin for 24 h, washed in PBS for 24 h, and embedded in paraffin.
RNA isolation and Northern blot analysis
Total RNA was isolated from CWR22 tumors as previously described
(21). Frozen tumors (100–200 mg) were pulverized in liquid nitrogen
and homogenized in 4 ml 4.0 M guanidine thiocyanate
(Fluka Chemical Co., Ronkokoma, NY) for 30–60 sec using a
homogenizer from Brinkmann Instruments, Inc. (model PT
10/35, Westbury, NY). Samples were centrifuged 10,000 x
g for 10 min at 4 C and the supernatants layered onto 1.25
ml cesium chloride cushions and centrifuged 36,000 x g
for 12 h at 4 C. RNA was dissolved in sterile H2O and
ethanol precipitated overnight at -20 C. RNA samples were resuspended
in sterile H2O, glyoxylated, fractionated by
electrophoresis in 1.0% agarose gels and transferred to Biotrans nylon
membranes (ICN Biomedicals, Inc., Aurora, OH).
complementary DNA (cDNA) probes were labeled with 32P-dCTP
(Amersham Corp., Arlington Heights, IL) using the
Prime-a-Gene System (Promega Corp., Madison, WI).
Membranes were hybridized in aqueous solution (5 x standard
sodium citrate (SSC), 5 x Denhardt’s solution, 1% SDS, and 100
µg/ml salmon sperm DNA) for 12 h at 68 C. After washing, the
membranes were exposed to x-ray film (Eastman Kodak Co.,
Rochester, NY) at -80 C with an intensifying screen. cDNAs for the
IGFBPs 1–6 were provided by Dr. S. Shimasaki (The Scripps Research
Institute, La Jolla, CA).
Quantitation of IGFBP-5 mRNA levels was performed by scanning Northern blots with an Ultroscan XL Laser Densitometer (LKB, Uppsala, Sweden). RNA loading differences were normalized by scanning the same blots subsequently hybridized with an 18S ribosomal RNA cDNA probe.
In situ hybridization histochemistry (ISHH)
ISHH was performed as described previously (22). Briefly,
dewaxed and rehydrated paraffin sections (6 µm) were treated with 0.2
N HCl, washed extensively with PBS, and hybridized with
biotin-labeled IGFBP-5 antisense riboprobe. The IGFBP-5 DNA
fragment corresponding to bp 558-1201 of rat IGFBP-5 cDNA (23) was PCR
amplified and cloned into pBlueScript (SK) vector
(Stratagene, La Jolla, CA). Biotin-labeled IGFBP-5
antisense riboprobe was generated using Genius RNA Labeling kit
(Boehringer Mannheim, Indianapolis, IN) and T3
RNA polymerase. The hybridization buffer contained 75% formamide, 10%
dextran sulfate, 3 x SSC, 50 mM sodium phosphate, pH
7.4, and approximately 1 ng/µl of probe. After incubation with probe
for 16–18 h at 55 C, slides were washed 4 times with 0.2 x SSC
for 1 h at 55 C. IGFBP-5 antisense riboprobe was detected with
monoclonal antibiotin antibody conjugated with horseradish peroxidase
(Boehringer Mannheim, 1:200) and diaminobenzidine
tetrachloride (DAB, Aldrich Chemical Co., Inc., Milwaukee,
WI). Rodent liver, a tissue lacking detectable IGFBP-5 mRNA (24 and our
unpublished observations), was used as a negative control.
Western immunoblot analysis
Lysates were prepared from frozen CWR22 tumors.
One-hundred-milligram tumor was pulverized in liquid nitrogen, allowed
to thaw on ice, and mixed with 1.0 ml RIPA buffer with protease
inhibitors (PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 0.5
mM phenylmethylsulfonyl fluoride, 10 µM
pepstatin, 4 µM aprotinin, 80 µM leupeptin,
and 5 mM benzamidine). Tissue was homogenized 30 sec on ice
using a Biohomogenizer (BioSpec Products, Inc.,
Bartlesville, OK) and incubated 30 min on ice. Samples were centrifuged
at 10,000 x g for 20 min at 4 C, supernatants
collected and centrifuged again. Aliquots of 100 µg supernatant
protein were electrophoresed on 12% SDS-polyacrylamide gels and
electroblotted to Immobilon-P membranes (Millipore Corp.,
Bedford, MA). Antihuman IGFBP-5 monoclonal antibody (Austral
Biologicals, San Ramon, CA) was used at 1:1000 dilution for
immunodetection. Human recombinant IGFBP-5 (Austral Biologicals) was
used as a positive control on immunoblots and ligand blots. Secondary
antibody (goat-antimouse IgG conjugated to horseradish peroxidase
(Amersham Corp., Arlington Heights, IL)) was detected by
enhanced chemiluminescence (DuPont NEN Research Products,
Boston, MA).
125I-IGF-I ligand blot analysis
For ligand blot analysis, 50–100 mg frozen tumor tissue was
pulverized, lysed, and sonicated for 30 sec in ice cold lysis buffer
(20 mM Tris-HCl, pH 7.4, 2% Triton X-100 and 10
mM EDTA) and centrifuged at 12,000 rpm in a microcentrifuge
for 5 min at 4 C. Aliquots of 100 µg supernatant protein were
separated by SDS-PAGE in 12% gels and electroblotted to nitrocellulose
membranes (Schleicher & Schuell, Inc., Keene, NH).
Membranes were incubated with hybridization buffer containing
125I-IGF-I (5 x 105 cpm/ml) as described
previously (25) for ligand blot analysis. Blots were exposed to Biomax
MS film (Eastman Kodak Co., Rochester, NY) with an
intensifying screen at -80 C for 2–4 days.
Immunohistochemical analysis
Formalin-fixed, paraffin-embedded sections of CWR22 tumors were
antigen retrieved by heating at 100 C for 30 min in a vegetable steamer
in the presence of antigen retrieval solution (CITRA, pH 6.0,
BioGenex Laboratories, Inc., San Ramon, CA) and cooled for
10 min. Slides were preincubated with 2% normal horse serum for 5 min
at 37 C and washed with automation buffer (Fisher Scientific International, Inc., Pittsburgh, PA). AR monoclonal antibody
F39.4.1 (BioGenex Laboratories, Inc.) was diluted 1:300
(0.13 µg/ml in PBS containing 0.1% BSA, pH 7.4) and reacted for 120
min at 37 C. Slides were incubated in biotinylated antimouse IgG
(Vector Laboratories, Inc., Burlingame, CA) for 15 min at
37 C (1:200 in PBS, pH 7.4) and in horseradish peroxidase-conjugated
avidin-biotin complex (Vector Laboratories, Inc.) for 15
min at 37 C (1:100 in PBS, pH 7.4). The immunoperoxidase complexes were
visualized using DAB for 8 min at 37 C (0.75 mg/ml in Tris buffer
containing 0.003% hydrogen peroxide, pH 7.6). Slides were
counterstained with hematoxylin (Gill’s formula, 1:6 dilution,
Fisher Scientific International, Inc.) for 12 sec.
Monoclonal antibody MIB-1 (Oncogene Science, Inc., Cambridge, MA) reacts with the cell-cycle-associated antigen Ki-67 that is highly expressed during the proliferative phases (G1, S, G2, and M) but is absent in the resting phase (G0) of the cell cycle. Ki-67 staining was performed at an IgG concentration of 0.5 µg/ml (1:50). All other steps were as described for AR immunostaining.
Formalin-fixed, paraffin-embedded sections (6 µm) of human prostate tissue were prepared for IGFBP-5 immunostaining with antihuman IGFBP-5 IgG monoclonal antibody (Austral Biologicals) using a 1:50 dilution (2 µg/ml in PBS). The antigen retrieval method was as described above for AR and Ki-67. Twenty tissue blocks each from benign prostatic hyperplasia (BPH) and androgen-dependent CaP and fifteen blocks from androgen-independent CaP were obtained for study. Tissue specimens of BPH and androgen-dependent CaP usually contained areas of prostatic intraepithelial neoplasia (PIN).
A second immunostaining procedure to identify basal epithelial cells was performed to distinguish between CaP and prostatic intraepithelial neoplasia (PIN) and BPH. Basal epithelial cells in all BPH and the majority of PIN specimens stain positive for the high molecular weight cytokeratins whereas CaP does not contain cytokeratin-positive basal cells (26). Following elution with glycine buffer (pH 2.3) to remove unreacted antibody, all slides were incubated with 1.7 µg/ml 34-ß-E12 anti-cytokeratin IgG monoclonal antibody (Enzo Diagnostics, Inc., Farmingdale, NY) for 15 min at 42 C. Antibody reaction was visualized using 3-amino-9-ethylcarbazole (AEC, BioGenex Laboratories, Inc.).
Quantitative analysis of IGFBP-5 immunostaining
Image acquisition. Imaging hardware consisted of a Zeiss
Axioskop microscope (Carl Zeiss, Inc., Thornwood, NY), a
3-chip CCD camera (C5810, Hamamatsu Photonics Inc., Hamamatsu, Japan)
and camera control board (Hamamatsu Photonics Inc.) and a Pentium-based
personal computer. Optical settings were calibrated using procedures
described previously (27). Images were sampled throughout histologic
sections but areas that contained necrosis, preparation artifacts or
the edges of sections were avoided. Three representative fields of view
were digitized for each histologic tissue type (as identified by a
pathologist): androgen-dependent CaP (n = 20),
androgen-independent CaP (n = 15), PIN (n = 20) or BPH
(n = 20) for a total of 75 prostatectomy specimens. Each field of
view for IGFBP-5-stained slides was digitized at total magnification
600x using a 20x dry objective (numerical aperture [NA] =0.6). A
digitized image consisted of 512 by 480 pixels in 16.7 million color
modes (but equivalent to 256 grayscale levels).
Digital image analysis. Immunopositive cells were recognized by intense cytoplasmic staining for IGFBP-5 in malignant glands and weak homogeneous staining with focal intense staining in PIN. An automatic image analysis algorithm was developed based on these immunostaining features. Segmentation of glandular areas was done using a convolution of Gaussian function with SD of 10 pixels and an adaptive local thresholding (28). Once the glandular area was segmented, a classification operation was performed to detect image pixels representing intense IGFBP-5 staining. Percent (% positivity) of the immunopositive area within the segmented glandular area was measured.
Statistical analysis. IGFBP-5 positive areas in BPH, PIN, and cancer specimens are expressed as the mean ± SE. Results were compared statistically by ANOVA. Significant differences among the means were determined using Fisher’s test with P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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). IGFBP-2, -3, -5, and
-6 mRNAs were detected as single bands of 1.8, 2.6, 6.0, and 1.4 kb,
respectively. IGFBP-1 mRNA was not detected, and IGFBP-4 mRNA gave
a weak hybridization signal. Of the IGFBP mRNAs expressed in CWR22,
only IGFBP-5 was androgen regulated. IGFBP-5 mRNA was decreased by 90%
at 6 and 12 days after castration of CWR22-bearing mice and treatment
with testosterone increased IGFBP-5 mRNA 10–12 fold within 24 h
(Fig. 2, A and B). Proliferation of tumor cells, measured by Ki-67
immunohistochemistry, was reduced in the 6-day castrate to 1% of the
intact, was increased 4-fold at 24 h and 40-fold after 48 h
of testosterone replacement (Table 1).
Cyclohexamide treatment (2.3 mg/mouse injected ip 3 h before tumor
removal) of 6 day castrated CWR22-bearing mice did not change the level
of IGFBP-5 mRNA, suggesting that message stability was not altered by
androgen withdrawal (data not shown). In CWR22 tumors that recurred
4–6 months after castration, IGFBP-5 mRNA was increased severalfold
higher than in CWR22 tumors from 6- and 12-day castrate mice and
similar to levels in CWR22 tumors from intact, androgen-stimulated
mice.
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). In tumors removed 6 days
after castration, relatively few epithelial cells were positive, and
immunostaining of the riboprobe was weaker (Fig. 3B). Treatment of 6
day castrate animals with testosterone propionate restored IGFBP-5 mRNA
in 90–95% of the epithelial cells within 24 h (Fig. 3C). In a
tumor that recurred several months following castration, IGFBP-5 mRNA
was detected in approximately 50% of the epithelial cells (Fig. 3D)
despite the absense of testicular androgen. Compared with tumor tissue
from the 6-day castrate mouse, both the number of cells expressing
IGFBP-5 mRNA and the intensity of the signal increased markedly.
In contrast to the epithelial cell expression of IGFBP-5 in CWR22, in
prostate specimens from patients with CaP, IGFBP-5 mRNA localized to
the stromal component in areas containing BPH and CaP (Fig. 4).
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). IGFBP-5
protein decreased following castration and increased in response to
androgen replacement at 6 and 12 days post castration. These changes
paralleled the androgen-regulated changes in mRNA expression described
above. Moreover, IGFBP-5 protein levels in the recurrent tumor were 3-
to 4-fold higher than in CWR22 tumors from 12-day castrate mice and
similar to levels in protein extracts of CWR22 tumors from the intact,
testosterone-stimulated mice. IGFBP-5 protein from CWR22 and recurrent
CWR22 bound 125I-IGF-I in Western ligand blot analyses
(Fig. 6) demonstrating the protein is
functional with respect to IGF-I binding. In the recurrent tumors,
compared with the androgen-stimulated tumors from intact mice, the
ratio of immunoreactive to ligand-bound IGFBP-5 appeared higher than in
CWR22 suggesting ligand-binding activity may be reduced in the
recurrent tumor.
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, C–F) compared with BPH and PIN (Fig. 7, A and B), (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In situ hybridization analysis confirmed the results obtained by Northern blotting and demonstrated uniform expression of IGFBP-5 mRNA in CWR22 epithelial cells. IGFBP-5 protein was expressed in parallel with mRNA and was active in binding 125I-IGF-I in ligand blot assays. However, the ratio of 125I-IGF-I bound relative to the amount of immunoreactive IGFBP-5 appeared lower in the recurrent tumor than in CWR22 from intact, androgen-stimulated mice. Immunohistochemical analysis of IGFBP-5 protein in human CaP tissue specimens demonstrated stronger immunostaining in CaP compared with BPH. The epithelial cell expression of IGFBP-5 mRNA and protein in CWR22 differed from expression in clinical CaP tissue samples where IGFBP-5 mRNA was predominantly in stromal cells. CaPs in general contain variable amounts of stroma, whereas CWR22 tumors are composed of xenografted epithelium and contain only small amounts of murine stroma. Nonetheless, in CaP tissue samples from patients as in CWR22, IGFBP-5 protein was associated with epithelial cells.
Our findings in CaP tissue samples from patients agree with results of an earlier study of radical prostatectomy specimens in which IGFBP-5 mRNA was higher in CaP than in BPH. IGFBP-5 mRNA localized to stromal cells surrounding acinar structures, whereas the protein was associated with cell membranes of epithelium and stroma (29). The increased epithelial cell expression of IGFBP-5 mRNA and protein in CWR22 suggests these cells have compensated for loss of human stroma by increasing expression of IGFBP-5 that could function to stimulate epithelial cells in an autocrine rather than a paracrine mode. The elevated IGFBP-5 expression in CaP glands compared with BPH implicates IGFBP-5 in the growth and progression of CaP. Figueroa et al. (30) found that mRNAs for IGFBP-5 and -2 were higher in CaP with higher Gleason scores while IGFBP-3 was lower, suggesting differential expression of these IGFBPs.
IGFBPs regulate target cell availability of IGFs for interaction with IGF receptors. Six IGFBPs have been described (IGFBP-1–6) (10, 12). IGFBP-1, -3, -4, and -6 inhibit IGF action in most systems studied, whereas IGFBP-2 and -5 can potentiate IGF-I functions. A carboxy-truncated form of IGFBP-5 stimulated mitogenesis of cultured osteoblasts (11). Thus, AR-stimulated expression of IGFBP-5 in CaP might enhance IGF-I action and tumor growth. IGFBP-5 binds specifically to plasminogen activator inhibitor-1 and to other components of the extracellular matrix in several cell and tissue types (31). Plasminogen activator-inhibitor-1 binding partially protects IGFBP-5 from proteolysis in vitro and may thereby regulate IGFBP-5 activity. The serine protease thrombin cleaves IGFBP-5 at physiological concentrations, thereby releasing IGF-I and increasing its bioavailability (32).
Previous studies on the regulation of IGFBP-5 have been largely
confined to cell lines. Both IGF-I and IGF-II increase IGFBP-5
production (33, 34, 35). In some cell-types, IGF increases secretion of
IGFBP-5 without detectable changes in mRNA (34, 35, 36, 37), whereas in other
cell types (33, 37), mRNA levels and secretion of IGFBP-5 are
increased. More recently, androgen was found to enhance the stimulatory
effect of IGF-I on IGFBP-5 mRNA and protein in cultured human genital
skin fibroblasts (38). Androgen induction was at the transcriptional
level as shown by nuclear run-on assays and analysis of IGFBP-5
promoter activity (39). Because the AR is also expressed in stromal
fibroblasts of human CaP, this observation suggests that androgen is
likely also to enhance the stromal cell production of IGFBP-5. Other
factors, however, have been reported to contribute to IGFBP-5
regulation, including cAMP (40), 1,25-dihydroxyvitamin D3,
PTH (35), interleukin-1
(36), transforming growth factor-ß (41),
and retinoic acid (40, 41). The human IGFBP-5 gene is on chromosome 2q
33–34 and consists of 4 exons. Promoter activity resides in the 461 bp
5' flanking region (42, 43). In cotransfection assays, Duan and
Clemmons (43) observed cell-type specific induction of promoter
activity with forskolin and found that AP-2 contributed to the cAMP
responsiveness of this gene.
In the recurrent CWR22 tumors, IGFBP-5 expression is increased through a mechanism that remains to be determined. We have identified several mRNAs that are up-regulated by androgen in the androgen-dependent CWR22, and these are also up-regulated in the recurrent tumor, whereas the levels of most mRNAs that are not androgen-regulated remain unchanged in the recurrent CWR22 (44). This observation suggests that androgen-regulated genes are up-regulated in the recurrent tumor by a common mechanism. Because AR expression is abundant in the recurrent tumor, one possibility is that AR becomes reactivated in the recurrent tumor despite the absence of testicular androgen. Androgen-independent activation of AR has been reported in CaP cell lines. Using PC-3 cells transiently cotransfected with AR and a reporter gene, Nazareth and Weigel (45) found that AR was transcriptionally activated by forskolin, an activator or protein kinase A. In similar assays using DU-145 cells, Culig et al. (46) reported that IGF-I alone stimulated AR-mediated gene transcription to the same extent as did the synthetic androgen methyltrienolone. In their study, IGF-I was a more effective activator of AR than either keratinocyte growth factor or epidermal growth factor. However, in CV-1 cells, Reinikainen et al. (47), using a different reporter vector and transfection method, found IGF-I and EGF increased AR transactivation only in the presence of androgen. Similarly, LNCaP cells required dihydrotestosterone in addition to IGF-I for growth stimulation.
IGF-I is known to stimulate CaP cell growth (4) and might be potentiated by IGFBP-5. Thus, androgen-stimulated IGFBP-5 could increase the growth effects of IGF-I in androgen-dependent CWR22. A similar effect could result from increased expression of IGFBP-5 in recurrent CWR22. Type I IGF receptor mRNA is expressed in both the androgen-dependent CWR22 and recurrent CWR22 tumors. The growth-promoting effect of IGF-I is modulated by IGFBP-3, which binds IGF-I and reduces its effect on epithelial cells. However, proteolysis of IGFBP-3 lowers its affinity for IGF-I (12). The IGFBP-3 proteases, prostate-specific antigen (PSA) and human kallikrien-2 (hK2) are AR-induced genes (48) that degrade IGFBP-3 (12, 49) and could thereby increase IGF-I stimulation of cell proliferation. PSA and hK2 are up-regulated by androgen in the androgen-dependent CWR22. Their expression is also increased in the recurrent CWR22 (44). Thus AR-induced increases in IGFBP-5, PSA, and hK2 might act in concert to potentiate IGF-I stimulation of cell growth both in androgen-dependent and androgen-independent CaP.
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Acknowledgments |
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Footnotes |
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Received September 2, 1998.
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in ovine articular
chondrocytes. Endocrinology 139:2356–2362, 25-dihydroxyvitamin D3 increases IGF
binding protein-5 expression in cultured osteoblasts. FEBS Lett 392:21–24[CrossRef][Medline]
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