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Geriatric Research Education and Clinical Center, Veterans Administration Puget Sound Health Care System (S.E.D., C.C.S., S.R.P.), Tacoma, Washington 98493; Department of Medicine, University of Washington (S.E.D., S.R.P.), Seattle, Washington 98195; Department of Pathology, Medical College of Virginia/Virginia Commonwealth University (C.D., J.L.W.), Richmond, Virginia 23298; and Department of Pediatrics, Oregon Health Sciences University (J.M.C., C.T.R.), Portland, Oregon 97201
Address all correspondence and requests for reprints to: Dr. Charles T. Roberts, Department of Pediatrics (NRC-5), Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97201. E-mail: robertsc{at}ohsu.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The P69SV40T series of PEC has been previously described with respect to tumor formation and the IGF system (9, 10, 11). The parental P69 cell line is poorly tumorigenic and not metastatic, whereas the M12 subline is greater than 90% tumorigenic and is metastatic (11). P69 cells express levels of IGF-IR similar to those seen in primary PEC. IGF-IR expression decreases significantly in the metastatic M12 subline, as has been reported in human metastatic prostate cancer and in murine prostate cancer models (5, 6, 10). The P69/M12 cell system thus represents an appropriate model in which to study the role and the regulation of the IGF-IR in the progression of prostate cancer.
When the IGF-IR is reexpressed in the malignant, metastatic M12 cell line to levels found in the benign parental P69 cell line, there is a concomitant decrease in tumor growth and metastasis in vivo, decreased growth in soft agar, and an increased sensitivity to apoptosis in vitro (12, 13). These data suggest that normal IGF-IR levels are necessary to maintain a less aggressive phenotype, and that altered responsiveness to IGF-I is alone sufficient to modulate the malignant phenotype.
The observation that both IGF-IR mRNA and protein decrease
significantly in the transition from benign to malignant human prostate
epithelium in vivo and in vitro is consistent
with the idea that this decrease is due to transcriptional regulation
(4). The transcription of the IGF-IR gene is controlled by
various factors, including Sp1 (14), p53 (15, 16), and WT1 (17, 18, 19, 20). The WT1 gene is located at
11p13 and encodes a DNA-binding protein with four
C2-H2 zinc finger domains
(21, 22). Multiple versions of the WT1 protein are
generated as a result of alternative splicing, RNA editing, and
alternative translation initiation codons (23, 24, 25). WT1
variants produced as a result of the alternative splicing of exon 9 are
designated WT1(+KTS) or WT1(-KTS) depending on the presence or absence
of a three-amino acid (Lys, Thr, Ser) insert between zinc fingers 3 and
4. The WT1 proteins arising from these transcripts appear to have
altered specificity for DNA binding and may regulate transcription in
different ways (26, 27, 28). The WT1 gene product belongs to
the early growth response family (29) and represses
the transcription of several growth factor and growth factor receptor
genes, including the IGF-IR (17, 18, 19, 20, 30). The IGF-IR
promoter contains 12 WT1 DNA-binding sites (18) in the
proximal 5'-flanking and 5'-untranslated regions (5'-UTRs; Fig. 1
) that contribute in an additive manner
to WT1 repression of IGF-IR promoter activity.
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In this study we have examined the level at which IGF-IR expression is regulated in metastatic and nonmetastatic prostate cancer cells and the potential contribution of WT1 to this regulation. We measured IGF-IR promoter activity in the benign P69 cell line (which expresses high levels of IGF-IR) and the malignant, metastatic M12 subline (which expresses low levels of IGF-IR) using IGF-IR promoter fragments to drive the expression of a firefly luciferase reporter gene. We also analyzed IGF-IR promoter activity and IGF-I-stimulated cell proliferation in stably transfected P69 cells overexpressing WT1(+KTS) to determine whether an increase in WT1 expression could influence IGF-IR expression and IGF responsiveness.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell lines and culture
Derivation of the P69SV40T (P69), and M12 cell lines has been
previously described (9, 10, 11). Briefly, human PEC were
immortalized with simian virus-40 T antigen to produce the immortalized
P69 cell line. P69 cells were injected sc into athymic nude mice and
produced tumor nodules in 2 of 18 animals after 180 days. These nodules
were reimplanted into athymic nude mice and after 3 passages resulted
in M12 cells, which demonstrated a short latency period of 7–10 days
to tumor formation in all 10 animals receiving sc injection and which
were locally invasive and metastatic when injected orthotopically
(11). All cells were cultured in RPMI 1640 medium
supplemented with 10 ng/ml EGF, 0.1 mM dexamethasone, 5
µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml selenium,
fungizone, and gentamicin (ITS medium) at 37 C in 5%
CO2. PC-3 and LNCaP cells were obtained from
American Type Culture Collection (Manassas, VA) and were
cultured in RPMI 1640 medium supplemented with 10% FBS. Human primary
PEC at passage 4 were obtained from Clonetics and seeded at 2.5 x
103 cells/T-25 flask in PrEBM supplemented with
bovine pituitary extract, insulin, hydrocortisone, GA-1000, retinoic
acid, transferrin, T3, epinephrine, and human EGF
supplied in a Bulletpack (Clonetics).
The B7 and D16 cell lines are stable clones of P69 cells transfected with a 3.0-kb human WT1(+KTS) cDNA cloned into the pcDNA3 mammalian expression vector (Invitrogen, San Diego, CA) (20). Clones were cultured in RPMI 1640 medium supplemented as described above and with 200 µg/ml G418. Transfectants were characterized for the expression of WT1 by RT-PCR and Western immunoblot, IGF-IR expression by Western immunoblot, and IGF-I-induced proliferation by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay.
Ribonuclease (RNase) protection assay of IGF-IR mRNA
levels
Primary human PEC, P69, M12, LNCaP, and PC3 cells were harvested
at 80% confluence, and total RNA was prepared using RNA-STAT60. RNA
quantity and integrity were verified by electrophoresis of 2-µg
aliquots on ethidium bromide-stained 1% formaldehyde-agarose gels,
followed by densitometric analysis using a Gel-Doc 1000 system and
Molecular Analyst software (Bio-Rad Laboratories, Inc.,
Hercules, CA). Twenty-microgram aliquots of total RNA were used in a
RNase protection assay with a [32P]UTP-labeled
antisense RNA probe complimentary to exon 2 and 3 sequences of the
human IGF-IR mRNA as previously described (17).
Hybridization signals were quantitated using a GS 375 phosphorimager
(Bio-Rad Laboratories, Inc.).
Cell surface IGF-IR levels
IGF-IR number per cell was determined by Scatchard analysis as
previously described (10). Briefly, cells plated in
24-well culture dishes were incubated in duplicate with
[125I]IGF-I (Amersham Pharmacia Biotech, Arlington Heights, IL) for 3 h at 26 C at
concentrations ranging from 0.05–2 nM. Nonspecific binding
was determined by adding a 500-fold molar excess of unlabeled IGF-I (a
gift from Eli Lilly & Co., Indianapolis, IN). After
incubation, the cells were placed at 4 C for 15 min and rinsed with
ice-cold PBS buffer containing 1% BSA. Cells were solubilized in 0.2
N NaOH, and the cell-associated radioactivity was counted
in a 
-counter. Protein was measured using the bicinchoninic acid
assay (Pierce Chemical Co., Rockford, IL), and cell number
was determined by counting cells in replicate wells. The maximum
binding capacity and apparent dissociation constant were determined
using Scatchard analyses as previously described (10).
Each experiment was performed in triplicate.
RT-PCR of WT1 mRNA
Total RNA was extracted from 20,000 cells using the Ultraspec
reagent and extraction protocol (Biotex Laboratories, Inc., Houston, TX). Total RNA was dissolved in
diethylpyrocarbonate-treated water containing RNasin (1 U/µl;
Promega Corp.) and stored at -80 C. One microgram of
total RNA was used in the RT reaction with 2 µM antisense
primer (5'-TCAAAGCGCCAGCTGGAGTTT-3'; corresponds to exon 10 of the WT1
gene), 75 mM KCl, 50 mM Tris (pH 8.3), 3.0
mM MgCL2, 0.8 mM
deoxy-NTPs, and 4.2 U/µl Moloney murine leukemia virus reverse
transcriptase (Life Technologies, Inc.) for 1 h at 42
C followed by heat inactivation at 95 C for 5 min. One tenth of the
first strand reaction was used in the amplification reaction. PCR was
performed in 56 mM KCl, 19.6 mM Tris (pH 8.3),
1.6 mM MgCl2, and 0.2 mM
deoxy-NTPs. The sense primer (5'-AGACATACAGGTGTGAAACC-3'; corresponds
to WT1 exon 8 sequence) and the antisense primer described above were
used at a final concentration of 1 µM and produced a
fragment of 225 bp. Forty cycles of PCR were performed using
denaturation for 1 min at 95 C, annealing for 30 sec at 60 C, and
extension for 1 min at 72 C using a GeneAmp 9600 thermal cycler
(Perkin-Elmer Corp., Foster City, CA). The PCR product was
analyzed by electrophoresis through a 1.5% agarose gel and transferred
to Zetaprobe (Bio-Rad Laboratories, Inc., Hercules, CA).
DNA was immobilized to the membrane with a UV cross-linker
(Stratagene, La Jolla, CA) and hybridized with the sense
primer that had been end-labeled with
[-32P]ATP (3,000 Ci/mmol; NEN Life Science Products, Boston, MA) at 55 C for 16 h.
Autoradiographs were developed after a 48-h exposure, and hybridizing
bands were quantitated by densitometry.
IGF-IR promoter reporter constructs
Transient transfection experiments used the following rat IGF-IR
gene promoter fragments ligated upstream of the firefly luciferase
reporter cDNA in pGL3 vectors: 1) fragment -476 to +641, designated
the full-length IGF-IR proximal promoter (20); 2) fragment
-476 to +41, designated the 5'-flanking region of the IGF-IR and
generated by PCR using a forward (5'-GGGCTAGCTTTTTCACAGAGCGGGCCAG-3')
and reverse (5'-GGAAGCTTCAAGAACCTCAGCCTCACCG-3') primer; and 3)
fragment +41 to +640, designated the 5'-UTR, generated by PCR using a
forward (5'-GGGCTAGCACGTGTGCGCGGCCCCGAGA-3') and reverse
(5'-GGAAGCTTCAGAAAGAGGAGCAAAGCCC-3') primer. Nucleotide +1 corresponds
to the transcription initiation site. All PCR primers were used at 5
µM. The forward primers of the 5'-flanking and 5'-UTR
fragments contained a NheI site, and the reverse primers
contained a HindIII site. Thirty-five cycles of PCR were
performed using a Gradient Cycler 40 (Stratagene). The
PCR-generated fragments were cut with NheI and
HindIII and ligated into the pGL3 reporter plasmid.
Constructs were verified by automated fluorescent sequencing. These
constructs are schematized in Fig. 1
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Transient transfections
P69, M12, D16, and B7 cells were transfected with the various
IGF-IR promoter/luciferase vectors using Tfx-50 according to the
manufacturer’s protocol. Briefly, cells were seeded in six-well plates
in complete RPMI medium containing 5% FCS and grown to 60%
confluence. Each well received 1.5 µg vector DNA using a 3:1 charge
ratio of Tfx-50/DNA in RPMI. After 1 h, the medium was replaced
with serum-containing medium, and the plates were incubated for 48
h, at which time the cells were lysed, and luciferase activities were
measured. All cells used in these experiments were
Mycoplasma free as determined by PCR with a
Mycoplasma PCR primer set (Stratagene).
Luciferase assays
Luciferase activity was determined using the Luciferase Assay
System (Promega Corp.) according to the manufacturer’s
protocol. Cells were lysed with 1 x reporter lysis buffer and
analyzed on a Nichols Institute Diagnostics
Luminometer 400 (San Juan Capistrano, CA). Each sample was read
for 20 sec. Transfection efficiency was determined by transfection of
duplicate wells with the pGL3 control vector in which luciferase
expression is driven by the simian virus 40 promoter as well as
cotransfection with a pCMV ß-galactosidase reporter plasmid as
previously described (20).
Western immunoblots
Cells were collected for Western blot analysis by washing with
PBS and 0.1% BSA. Cells were lysed in 100 µl SDS sample buffer (10%
glycerol, 2% SDS, and 0.001% bromophenol blue) and heated for 5 min
at 100 C. Electrophoresis of cell lysates was performed on a 10%
SDS-PAGE gel. Protein concentration was determined with the
bicinchoninic acid protein assay reagent, and 50 µg protein were
added to each lane. After electrophoresis, proteins were transferred to
nitrocellulose by electroblotting and probed with either IGF-IR or WT1
antibodies. Bound antibody was detected using ECL (Amersham Pharmacia Biotech, Piscataway, NJ). Buffers and wash times were
as suggested in the ECL kit, except that nitrocellulose was washed in
10% hydrogen peroxide for 10 min before the first blocking step.
Assays of tumor growth in vivo
The tumorigenicity of P69, M12, and PC-3 cell lines was assessed
by sc injection of 106 cells into male athymic
nude mice that were 6–8 weeks old at the time of injection. Each group
of 10 mice was injected with 1 of the 3 cell lines. The percentage of
tumor formation was defined as the percentage of mice injected with
tumor cells that developed tumor nodules over the 12-week period
postinjection. All mice were maintained in a specific pathogen-free
barrier facility. In vivo experiments were conducted under a
protocol approved by the VAPSHCS and University of Washington
animal care and use committees.
Cell proliferation
Cell proliferation was determined with a tetrazolium
assay for quantification of viable cells (Cell Titer 96 Aqueous kit,
Promega Corp.). Twenty-five hundred cells were added to
each well of a 96-well plate, and test reagents were added at the times
indicated. IGF-I was added to the ITS medium at the time of plating.
After 72 h in culture, the tetrazolium salt and dye solution was
added, color development was allowed to proceed for 2–3 h at 37 C, and
absorbance at 570 nM was measured for each well. Each cell
line was tested in three separate experiments. MTS results were
confirmed by cell counts. The correlation between cell number and the
MTS tetrazolium assay in our laboratory is r = 0.97.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Specifically, the M12 and PC-3 cell
lines are highly tumorigenic and metastatic and display the lowest
levels of IGF-IR protein and mRNA. The poorly tumorigenic P69 cell line
and primary PEC express high levels of IGF-IR protein and mRNA. LNCaP
cells, which are weakly metastatic, exhibit intermediate levels of
IGF-IR expression. No changes in affinity for IGF-I were seen.
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, the
activity of all three constructs was significantly lower in M12 cells
than in P69 cells (50%, P < 0.01; 53%,
P < 0.0001; and 36%, P < 0.01,
respectively).
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).
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). Overexpression of WT1(+KTS) resulted
in a marked decrease in IGF-IR protein in the B7 and D16 clones to
levels comparable to those seen in the metastatic M12 cells. The
decreased level of IGF-IR in the B7 and D16 clones resulted in a
decreased proliferative response to IGF-I compared with that seen in
the parental P69 cell line (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The coordinate decrease in IGF-IR protein or binding and mRNA levels seen in metastatic prostate cancer cells is consistent with the idea that transcriptional repression of the IGF-IR gene is a requisite for the development of advanced disease. In that case, an understanding of the molecular mechanisms governing transcription of the IGF-IR gene in PEC would provide insights into improved approaches for the treatment of refractory disease. The results of the current study demonstrate that IGF-I promoter activity itself is decreased in metastatic prostate cancer cells, and that this decrease is responsible for a substantial portion of the decreased IGF-IR mRNA and protein observed in these cells compared with their nontumorigenic or metastatic precursors. That the difference in IGF-IR mRNA levels between P69 and M12 cells is greater than the difference in promoter activity suggests that regulatory elements outside of the -476/+640 proximal promoter region analyzed in this study may influence the activity of the endogenous IGF-IR gene in the P69-M12 cell system. Alternatively, posttranscriptional regulatory mechanisms may also contribute to overall IGF-IR gene regulation in this context. Our data, however, suggest that transcriptional control is a principal component of IGF-IR gene expression in metastatic vs. nonmetastatic prostate cancer cells.
We have previously shown that IGF-IR gene expression is significantly increased in Wilms’ tumor, and that there is a negative correlation between levels of IGF-IR mRNA and WT1 mRNA, suggesting that the IGF-IR is under the inhibitory control of the WT1 tumor suppressor protein (17). Characterization of the IGF-IR promoter using gel retardation and deoxyribonuclease I footprinting assays demonstrated 12 WT1-binding sites in the -476/+640 region of the proximal IGF-IR promoter (18). Overexpression of WT1 in Chinese hamster ovary and G401 cells resulted in decreased IGF-IR promoter activity and cell surface receptor number (18, 20).
Overexpression of WT1(+KTS) in P69 cells resulted in highly significant suppression of IGF-IR promoter activity with all three promoter constructs. Interestingly, promoter activity in the B7 clone was suppressed to the same extent as in the D16 clone, yet the levels of WT1 expression were higher in B7 cells compared with D16 cells. This may indicate that even moderate increases in WT1(+KTS) can efficiently repress IGF-IR gene expression. The effect of WT1 on IGF-IR promoter activity was paralleled by decreased expression of the endogenous IGF-IR gene and a decreased proliferative response to IGF-I. A short WT1 transcript has been recently identified in some prostate cancer cell lines that lacks the sequence derived from the first five exons (38). It remains to be determined whether the protein encoded by this novel WT1 transcript has altered binding specificity or function in prostate cells, but our previous demonstration that carboxyl-terminal versions of WT1 are functional (20) suggests that this WT1 variant may also repress IGF-IR gene expression.
In summary, this study demonstrates that the decrease in IGF-IR expression that occurs in aggressive prostate cancer appears to be primarily at a transcriptional level, and that an increase in WT1 action may constitute part of the mechanism through which IGF-IR gene expression is repressed. The level of IGF-IR expression may, in turn, affect IGF responsiveness in terms of proliferation, differentiation, or sensitivity to apoptosis. Any or all of these factors could influence the tumorigenecity of a given PEC. In light of our previous studies demonstrating decreased tumor growth and metastatic potential as a result of increased IGF-IR expression (12, 13), targeting those factors that regulate IGF-IR promoter activity may lead to novel therapies for metastatic disease.
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Footnotes |
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Received June 23, 2000.
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-subunit of the IGF-IR. Each
lane contains 100 µg protein. Note the marked decrease in IGF-IR
expression in the metastatic M12 line and the B7 and D16 clones,
derived by transfection of the P69 line with a WT1 expression vector,
compared with the P69 parental line.