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Reproductive Endocrinology |
Children’s Hospital of Philadelphia, University of Pennsylvania, (G.D., R.R., P.C.), Philadelphia, Pennsylvania 19104; Departments of Urology (D.M.P.) and Medicine (A.R.H., T.V.), Stanford University Medical Center, Palo Alto, California 94305; and Department of Pediatrics, Oregon Health Sciences University (C.T.R., R.G.R.), Portland, Oregon 97201
Address all correspondence and requests for reprints to: Pinchas Cohen, Pediatric Endocrinology, Children’s Hospital of Philadelphia, University of Pennsylvania, Room 410-D Abramson Research Center, 3400 Civic Center Boulevard, Philadelphia, Pennsylvania 19104. E-mail: cohenp{at}email.chop.edu
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Abstract |
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Introduction |
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The insulin-like growth factor (IGF) axis is a multicomponent network of molecules involved in the regulation of cell growth. This axis includes the ligands IGF-I and IGF-II (7), cell-surface receptors, which include the type 1 IGF receptor (IGF-1R) (8), and a family of high-affinity binding proteins (IGFBPs), which regulate IGF availability to the receptors (9, 10). The IGFs, their receptors, and their binding proteins participate in endocrine as well as autocrine-paracrine growth processes and may be involved in neoplastic transformation (7, 8, 9, 10).
The IGF-1R is a membrane-bound tyrosine kinase that mediates the trophic, metabolic, and differentiative effects of the IGFs (11, 12). Overexpression of IGF-1R in Balb/c 3T3 cells has been shown to abrogate all requirements for exogenous growth factors, suggesting that this receptor plays a central role during the cell cycle (13). Furthermore, the constitutive expression of IGF-1R gene in most tissues is consistent with the putative role of IGFs as critical regulators of cellular growth and differentiation (14).
The Wilms’ tumor gene, WT-1, encodes a zinc-finger DNA-binding protein that has been shown to be deleted or mutated in renal malignancies such as Wilms’ tumor (15). This gene has also been shown to be critical for mesonephric development (16). The role of WT-1 as a tumor suppressor gene has been demonstrated in Wilms’ tumor-derived cell lines in which the WT-1 gene has been shown to be mutated. After transfection with a wild-type WT-1 expression vector, a loss of the malignant phenotype was observed (17). WT-1 has been shown to repress the expression of a number of genes including Igf2 and Igfr (18, 19), as well as those encoding other growth factors and receptors (20). It has been suggested that the loss of negative regulation of the IGF-1R and IGF-II in Wilms’ tumor is the primary process that mediates tumor progression (19, 21).
We have investigated the role of the IGF axis in the prostate by documenting the presence of IGFs and their receptors and IGFBPs and their proteases in seminal plasma (22, 23, 24) and in cultured prostatic cells (25, 26, 27). We have further shown that prostatic stromal cell strains from BPH patients demonstrate increased IGF-II expression and altered protein and messenger RNA (mRNA) expression of IGFBP-2 and IGFBP-5 (28, 29). We then proposed that IGF axis abnormalities may be involved in the pathogenesis of BPH (25, 26, 27, 28, 29).
Hypothesizing that disregulated IGF-1R and IGF-II mRNA expression in the prostates of patients with BPH may be related to altered WT-1 status, we have evaluated WT-1, IGF-1R, and IGF-II gene expression in prostatic cells from such patients and controls.
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Methods |
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Tissue samples were dissected from histologically normal peripheral or central zones, BPH, or adenocarcinomas of specimens obtained by radical prostatectomy, cystoprostatectomy, or suprapubic enucleations from men 55–80 yr old after obtaining informed consent (approved by the Stanford University Institutional Review Board). There was no statistical difference in the mean ages of the three groups studied. None of the patients had received any prior hormonal, radiation, or chemical therapy. Following overnight collagenase digestion of the tissues, epithelial cultures were derived according to previously published protocols (30). Primary and serial cultures of epithelial cells were maintained in serum-free medium (PFMR-4A supplemented with growth factors) (30).
Stromal cell strains were established by inoculating collagenase-digested tissues into MCDB 105 (Sigma, St. Louis, MO) supplemented with 10% FBS and gentamicin (100 µg/ml) (31). Cells were serially passaged in this same medium. Stromal cell cultures were not labeled by antibodies against keratins or factor VIII, but were stained by antibodies against vimentin and fibronectin.
Cells were grown in serum-free media for 72 h and then used for RNA analysis. Twenty five different stromal strains \[9 normal, 9 BPH, 7 derived from prostatic adenocarcinoma (CaP)\] and 17 different epithelial strains (7 normal, 5 BPH, 5 CaP) were used for the various analyses. Cells were studied between passage 10 and 15.
RNA analysis
Total RNA was isolated from freshly lysed cells as previously described (24). RNA samples (1 µg) were analyzed by quantitative RT-PCR as previously described (32). PCR was performed on a Perkin Elmer 4800 thermocycler, and all RT-PCR reagents were purchased from Perkin-Elmer/Cetus (Norwalk, CT).
After RT, complementary DNA (cDNA) was amplified with the following WT-1 primers: sense, 5'-CTG GAA TCA GAT GAA CTT AGG-3' and antisense, 5'-ACC TGT ATG AGT CCT GGT G-3'. These primers amplify a 461-bp double-stranded DNA sequence that flanks exon 5. PCR conditions were 94 C for 1 min, 60 C for 1 min, and 72 C for 1 min for 30 cycles. IGF-II primers were: sense, 5'-GGG AAT TCA TTG CTG CTT ACC GCC CCA G-3' and antisense, 5'-GGA AGC TTA GTA CGT CGT CTC CAC GAG GGC C-3'. These primers amplify a 200-bp double-stranded DNA sequence. PCR conditions were as above, except that the denaturing temperature was 93 C instead of 94 C and 25 cycles were used. RNA quantity was normalized for 18S ribosomal RNA (32) that was amplified using the same protocol for 20 cycles after optimization of the assay indicated that to be the ideal linear range for this high abundance ubiquitous mRNA.
The PCR products were electrophoresed on an ethidium bromide-stained 2% agarose gel (GIBCO BRL Ultra Pure, Gaithersburg, MD) in TAE buffer. Gels were photographed and analyzed densitometrically on a BioRad 670GS scanning densitometer (Hercules, CA).
For IGF-1R RNA analysis, the same thermocycler and reagents were used, and 33P-labeled deoxycytidine ATP was purchased from Amersham (Arlington Heights, IL). The primer set for human IGF-1R mRNA was: sense, 5'-GGG AAT TCC CCG ACC TCG CTG TGG GG-3' and antisense, 5'-GGA AGC TTG GAA CAG CAG CAA GTA CTC-3'. These primers amplify 255-bp spanning nucleotide positions 64–310 (33). PCR conditions for the above primers were 94 C for 1 min, 62 C for 1 min, and 72 C for 1 min for 30 cycles. RNA quantity was normalized for human ribosomal protein 7 (hL7) mRNA (26), which was amplified by the same protocol but for 15 cycles. The PCR samples were run with [33P deoxycytidine triphosphate (1 million cpm in 100 µl reaction mixture), and the reaction products were separated in a precast 10% polyacrylamide TBE minigel (Bio-Rad). Gels were exposed to film overnight and analyzed by densitometry using Molecular Analyst Version 2.0 software (Bio-Rad, Hercules, CA). The sequences of the genes studied by RT-PCR were obtained from Genebank. The primers for PCR were designed by the Macvector software (Oxford Molecular, Oxford, United Kingdom).
Solution-hybridization/RNase protection assay
mRNA identification using 32P-labeled antisense RNA probes for WT-1, IGF-1R, and cyclophilin were performed on samples as previously described (19, 21). Briefly, 20-µg aliquots of total RNA were hybridized overnight with the probes. After RNase digestion, protected hybrids were resolved on denaturing 6% acrylamide gels, exposed to film, and analyzed by densitometry. To generate a WT-1 antisense RNA probe, a 244-bp BstXI-BamHI fragment corresponding to exon 1 and 2 sequences of a WT-1 cDNA was cloned into pGEM3Z using a single-stranded linker-adapter oligonucleotide. This construct was linearized with HindIII and transcribed with T7 RNA polymerase to produce a 269-base probe, 244 bases of which were complementary to WT-1 mRNA. A human cyclophilin probe template for in vitro transcription was purchased from Ambion (Austin, TX).
Statistical analysis
Experiments were repeated three to five times. Densitometric data were analyzed using standard statistical methods, including Student’s t test and ANOVA. Results are reported as mean \ SEM.
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Results |
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We evaluated WT-1 expression in cultured prostatic cells of different histological origins. Total RNA from normal (n = 9) and BPH (n = 9) stromal cell strains was extracted as described above and used for WT-1 mRNA analysis by RT-PCR. When compared with normal stromal cell strains, BPH stromal strains showed a dramatic decrease in WT-1 mRNA expression.
As seen in Fig. 1A
, WT-1 mRNA in normal strains appeared
as a double band representing the two normally occurring alternatively
spliced variants of the WT-1 transcript that differ because of the
presence or absence of 51 bases within exon 5 (17). Five normal samples
(lanes 7–11) and five BPH samples (lanes 2–6) are shown in respect to
the molecular weight markers (
x 174/HaeIII ladder and
1-kilobase DNA ladder). Of note, normal strains exhibited some
variability in the degree of WT-1 mRNA expression. In comparison, the
WT-1 expression in BPH stromal cells was very low or undetectable in
all of the nine strains examined.
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. After normalization for 18S RNA, stromal
strains from BPH exhibited a 7-fold decrease in WT-1 mRNA relative to
normal strains (P < 0.01).
Analysis by solution hybridization/RNase protection assay corroborated
this decrease in WT-1 mRNA levels. As shown in the autoradiograph of
Fig. 2, a 240-base protected probe fragment
corresponding to the WT-1 mRNA was clearly seen in five normal stromal
strains (lanes 2–6), but was essentially undetectable in three BPH
strains (lanes 7–9). The identity of the minor band of approximately
60 bases is unknown, but was WT-1 specific and may represent an
alternatively spliced minor mRNA form.
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).
Analysis of stromal strains derived from prostatic adenocarcinoma
disclosed amounts of WT-1 mRNA similar to those detected in normal
strains (Table 2).
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IGF-1R expression was evaluated from similar prostatic cell strains. Total mRNA from normal and BPH stromal cell strains was extracted as described above and used for IGF-1R mRNA analysis by 33P-labeled RT-PCR. When compared with normal stromal cell strains, BPH stromal strains showed an increase in IGF-1R mRNA expression.
As seen in Fig. 3A, the RT-PCR product derived from
IGF-1R mRNA in normal strains appeared as a single distinct band. Seven
normal samples (lanes 2–8) and seven BPH samples (lanes 10–16) are
shown with respect to the molecular weight markers (x
174/HaeIII ladder). Densitometric analysis of these bands
was normalized to that of the L7 housekeeping gene for each sample and
then plotted.
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. After normalization for hL7 mRNA, stromal
strains from BPH exhibited a 3-fold increase in IGF-1R mRNA relative to
normal strains (P < 0.05). Increased IGF-1R mRNA in
prostatic stromal cells from BPH patients was verified using RNase
protection assays (data not shown).
Analysis of prostatic epithelial cells disclosed no change in
expression of IGF-1R mRNA in hyperplastic strains (Table 1). Analysis
of stromal strains derived from prostatic adenocarcinoma disclosed
amounts of IGF-1R mRNA similar to those detected in normal strains
(Table 2).
IGF II mRNA expression in cultured prostatic cells
Total RNA from normal and BPH stromal cell strains was extracted as described above and used for IGF-II RNA analysis by RT-PCR. When compared with normal stromal cell strains, BPH stromal strains showed a dramatic increase in IGF-II mRNA expression.
As seen in Fig. 4A, the RT-PCR product derived from
IGF-II mRNA in normal strains appeared as a single distinct band. Three
normal samples (lanes 2–4) and three BPH samples (lanes 5–7) are
shown with respect to the molecular weight markers in lanes 1 and 8
(x 174/HaeIII ladder and 1-kilobase DNA ladder). The
bands representing the PCR product of IGF-II were dark in the BPH
samples and were faint or almost invisible in the normal samples,
indicating increased expression of IGF-II mRNA in BPH.
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. After normalization for h18S RNA, stromal strains from BPH
exhibited a 10-fold increase in IGF-II mRNA relative to normal strains
(P < 0.01). Analysis of IGF-II expression by Northern
blotting analysis disclosed a similar increase in IGF-II expression in
hyperplastic strains, as we have previously demonstrated (28) (data not
shown).
Analysis of prostatic epithelial cells revealed no expression of IGF-II
mRNA in either normal or hyperplastic strains (Table 1) when tested by
Northern blotting. Analysis of stromal strains derived from prostatic
adenocarcinoma disclosed amounts of IGF-II mRNA similar to those
detected in normal strains (Table 2).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The IGF-1R is a transmembrane tyrosine kinase that mediates the trophic, metabolic, and differentiative effects of IGF-I and IGF-II. Beginning at early organogenesis, the IGF-1R gene is constitutively expressed by most body tissues, consistent with the role of the IGF axis in mediating proliferation and differentiation (12, 13, 14, 15, 34). Several studies have shown that the overexpression of IGF-1R in a fibroblast cell line abrogates all requirements for exogenous growth factors, suggesting that this receptor mediates a central mechanism in cell cycle (13).
Although relatively little is known about the molecular regulation of the IGF axis in the prostate, it has been shown in other tissues that several transcription factors are known to control the expression of IGFs and the IGF-1R. Among these are SP1, AP1, P53, CEBP (7, 8), and WT-1 (15, 20). WT-1 is an inhibitory transcription factor that affects multiple genes, of which the most important may be IGF-II and IGF-1R (15, 16, 17, 18, 19, 20, 21). WT-1 also suppresses the expression of other growth-regulatory genes, including platelet growth factor-A derived (PDGF-A) (15, 20), transforming growth factor-1 (35), colony-stimulating factor-1 (36), and the retinoic acid receptor (37).
WT-1 has been previously shown to be involved in mesonephric development and differentiation. In humans, germline mutations of WT-1 are associated with urogenital malformations as well as Wilms’ tumors (15, 20). Transgenic mice homozygous for targeted disruption of WT-1 die in utero and fail to develop kidneys and gonads (16).
At least in Wilms’ tumors, it appears that the increase in the autocrine effects of IGF-II on the IGF-1R is the most critical abnormality that results from decreased WT-1 suppression of gene expression. Supporting this statement is the fact that virtually identical phenotypes may be observed in Wilms’ tumors associated with WT-1 mutations and in those associated with IGF-II gene duplication or loss of imprinting of IGF-II (38).
In this study, we demonstrated that the proliferative disorder BPH is characterized by a decrease in WT-1 mRNA associated with elevated expression of IGF-1R and the IGF-II in prostatic stromal cells. This association is compatible with previous reports demonstrating that WT-1 inhibits transcription of the promoters for the IGF-1R (19, 39) and IGF-II genes (40). It has also been shown that transfection of WT-1 into Wilms’ tumor cells concomitantly suppresses growth as well as IGF-II and IGF-1R expression (21).
BPH is an extremely common disorder, affecting a large proportion of elderly men (1, 2). The etiology of this condition remains unknown, but it appears to be related to local factors rather than to systemic hormonal changes. The trophic systems that may influence prostate growth, such as the androgenic and somatotrophic pathways, normally operate at a reduced, rather than increased tone at the age during which BPH develops. Histologically, the changes that occur in the hyperplastic prostate are reminiscent of a regression to a fetal-like state, and include a proliferation of the stroma followed by epithelial growth (3, 4). To date, no specific molecular defects have been described that could explain these changes. The production of a locally active growth factor that would mediate these phenomena has been proposed (5, 6), and several candidate hormones have been suggested. The loss of WT-1 in the prostatic stroma is compatible with the above hypothesis. Our finding of increased IGF-II and IGF-1R expression in cultured cells could well be related to increased proliferation in vivo.
WT-1 has been implicated in several human diseases other than Wilm’s tumor. Testicular cancer has been demonstrated to have decreased expression of WT-1 (15, 20). On the other hand, the WT-1 gene was found to be overexpressed in several tumor types including leukemia (41) and ovarian cancer (42), where its mRNA levels appeared to be much higher than in normal tissues. These phenomena may be related to WT-1’s ability to enhance transcription of growth factors and other genes when present in a mutated form (17) or in the presence of modulating factors (15).
A role for WT-1 in prostate disease has not previously been suggested, but our findings indicate that WT-1 may regulate IGF axis homeostasis within the prostatic stroma, and that BPH is associated with a loss of WT-1 and the resulting up-regulation of the IGF-II and the IGF-1R leading to cellular hyperplasia. We have found WT-1 to be reduced in primary cultures from multiple patients, giving further credence to its potential role in the etiology of BPH. It is as yet unclear what mediates the reduction of WT-1 expression in BPH. It may be postulated that its own transcription is regulated by some yet undefined factor that rises with advancing age.
The potential speculative relation between altered WT-1, IGF-1R, and
IGF-II expression and hyperplastic prostate growth is shown in a
schematic fashion in the theoretical cartoon in Fig. 5.
In normal prostatic stromal cells the levels of WT-1 expression
maintain the transcriptional levels of IGF-1R and IGF-II and thereby
regulate the rate of stromal cell proliferation. In BPH, repressed
transcription of WT-1 in the stromal cells could be associated with
increased IGF-1R and IGF-II transcription. This presumably increasingly
available IGF-II binds to IGF-1R and initiates its mitogenic effect on
prostate stromal cells, thus, theoretically, contributing to the
pathogenesis of BPH.
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Acknowledgments |
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Footnotes |
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Received January 21, 1997.
Revised March 21, 1997.
Accepted April 3, 1997.
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and nine BPH 
cell
strains; mean ± SEM of each is also shown.
and 7 BPH • strains is shown; mean ±
SEM of each is also shown.
