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Department of Pediatrics (A.L.-B., C.K.B., G.R.D., V.H., Y.O., R.G.R.), Oregon Health Sciences University, Portland, Oregon 97201; and Geriatric Research Education and Clinical Center (S.R.P.), Veterans Affairs Health Care System Puget Sound, Seattle/Tacoma, Washington 98493
Address all correspondence and requests for reprints to: Ron G. Rosenfeld, M.D., Department of Pediatrics, School of Medicine, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97201-3098. E-mail: rosenfer{at}ohsu.edu
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Abstract |
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IGFBP-rP2 (also known as connective tissue growth factor), a downstream effector of transforming growth factor (TGF)-ß and modulator of growth for both fibroblasts and endothelial cells, was detected in most of the normal and malignant prostatic epithelial cells tested, with a marked up-regulation of IGFBP-rP2 during senescence of HPEC. Moreover, IGFBP-rP2 noticeably increased in response to TGF-ß1 and all-trans retinoic acid (atRA) in HPEC and PC-3 cells, and it decreased in response to IGF-I in HPEC.
IGFBP-rP3 [nephroblastoma overexpressed (NOV)], the protein product of the NOV protooncogene, was not detected in HPEC but was expressed in the tumorigenic DU145 and PC-3 cells. It was also synthesized by the SV40-T antigen-transformed P69 and malignant M12 cells, where it was down-regulated by atRA.
These observations suggest biological roles of IGFBP-rPs in the human prostate. IGFBP-rP1 and IGFBP-rP2 are likely to negatively regulate growth, because they seem to increase during senescence of the prostate epithelium and in response to growth inhibitors (TGF-ß1 and atRA). Although the data collected on IGFBP-rP3 in prostate are modest, its role as a growth stimulator and/or protooncogene is supported by its preferential expression in cancerous cells and its down-regulation by atRA.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Abnormalities in the IGF system have been identified in prostate disease, such as prostate hyperplasia and cancer. In this respect, prostate cancer growth seems to be poorly dependent on IGFs, because the type I IGF receptor, which mediates most biological functions of IGF-I and IGF-II, is down-regulated during prostate carcinogenesis (3). Additionally, recent epidemiological studies have shown an increased risk of developing prostatic carcinoma in adult males with high-normal serum concentrations of IGF-I (4).
Despite extensive characterization of the IGFBPs in the human prostate, little is known about their roles in prostate physiology. IGFBP-2 and -4 are known to increase during carcinogenesis of the prostatic epithelium (5, 6), whereas IGFBP-3 is a proteolytic substrate for prostate-specific antigen (7, 8). Recent studies have also shown an increase of several IGFBPs in prostate tissue during involution of the gland in castrated rats and in men taking finasteride (9, 10). These findings suggest that IGFBPs may play a role in apoptosis of prostate cells, either by sequestering IGFs or by direct cellular actions. Our knowledge of the new IGFBP-rPs in the human prostate is only scant at its best.
The IGFBP-rPs are cysteine-rich proteins involved in a diversity of biological functions, including growth regulation. IGFBP-rP1 was originally cloned from leptomeningial cells and was termed meningioma-associated complementary DNA (cDNA) (MAC25) (11). This protein has also been reported as tumor-derived adhesion factor (TAF, recently renamed Angiomodulin) (12, 13), prostacyclin-stimulating factor (14), and T1A12 (15). In the human prostate, we have also recently described preferential expression of IGFBP-rP1 in normal human prostate cells and tissues, compared with their malignant counterparts, and we have shown that this protein is up-regulated by transforming growth factor (TGF)-ß1 and all-trans retinoic acid (atRA) in prostate epithelial cells (16).
IGFBP-rP2 [also known as connective tissue growth factor (CTGF)] was initially isolated from human umbilical endothelial cells and shown to be mitogenic and chemotactic for fibroblasts (17). IGFBP-rP2 belongs to the CCN [for CYR61, CTGF, and nephroblastoma overexpressed (NOV)] family of cysteine-rich proteins involved in a diversity of cellular functions, such as mitogenesis, differentiation, survival, adhesion, migration, and regulation of matrix gene expression (18). IGFBP-rP2 is also a major downstream effector of TGF-ß in fibroblasts, where it was evidenced that TGF-ß controls IGFBP-rP2 expression via a novel response element in the IGFBP-rP2 gene (19). To date, however, no studies have been reported on the role of IGFBP-rP2 in prostate biology and/or carcinogenesis.
IGFBP-rP3 (also known as NOV) was first recognized as an aberrantly expressed gene in avian nephroblastoma and later shown to be also overexpressed in the human homologue Wilms tumor (20, 21). In the human prostate, it has been reported that NOV is differentially expressed in PC-3 cells, compared with other cancerous and normal cells in culture, but the significance of this finding is unknown (22).
We now report that prostate epithelial cells, in culture, express not only IGFBP-rP1 and 3, but also IGFBP-rP2, and that these proteins are responsive to growth regulators. Interestingly, IGFBP-rP1 and 2 also increase during senescence of normal prostate epithelial cells, thus supporting growth-regulatory roles of these proteins in the prostatic epithelium.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
Figure 1
summarizes the cell lines
employed in these studies. HPEC cells were maintained in PrEBM media
supplemented with the following: bovine pituitary extract (BPE),
insulin, hydrocortisone, GA-1000, retinoic acid, transferrin,
levothyroxine, epinephrine, and human EGF. HPECs were subcultured as
recommended by the manufacturer. When they reached 80% confluence, all
growth factors were withdrawn, except for BPE, for 12 h. Medium
was changed again to PrEBM plus BPE for cell proliferation and growth
factor regulation studies.
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Cellular proliferation assays
Cells were seeded at a density of 1 x
104 cells per well, in 48-well plates (Falcon,
Becton Dickinson and Co. Labware, Franklin Lakes,
NJ), in 500 µl of either rich defined medium or 10%
FBS-enriched medium, depending on the cell line. When 80% confluence
was reached, the medium was changed to either basal medium or serum
free medium for 12 h and then treated with various doses of
TGF-ß1 (0–5 ng/ml), atRA (0.001–1 µM), or IGF-I
(0–100 ng/ml) in 250 µl of the same serum-free medium for 48 h.
[3H]thymidine (0.4 µCi/ml) was added for the
last 24 h (HPEC and PC-3 cells) or for the last 6 h (P69 and
M12 cells). After labeling, the cell layers were washed twice with PBS
and incubated with 10% trichloroacetic acid (TCA) at -20 C for 15
min, followed by another wash with 10% TCA before the cells were lysed
with 0.25 N sodium hydroxide, and the precipitated material
was read by means of an LS 6500 Scintillation Counter (Beckman Coulter, Inc., Fullerton, CA).
Cell proliferation was also tested by MTS assay kit, according to the manufacturer’s instructions. Cells were incubated for the last 2 h of treatment, in the presence of the MTS reagent, and absorbance was measured at A490 nm.
Growth factor regulation studies
Cells were seeded at a density of 10 x
104 cells per well, in 60-mm tissue culture
dishes, and grown to 80% confluence. Treatments with TGF-ß1 (0–5
ng/ml), atRA (0.001–1 µM), or IGF-I (0–100 ng/ml) were
also done under serum-free conditions, for 48 h, after which
conditioned media, total cell lysates, and total cytoplasmic RNA were
collected for Western immunoblots (see Western immunoblot
analyses) and Northern blots (see RNA analyses)
studies.
Western immunoblot analyses
Conditioned media and total cell lysates, using RIPA buffer
[150 mM NaCl, 20 mM HEPES (pH 7.4), 1%
(vol/vol) Triton X-100, 1% (wt/vol) sodium deoxycholate, 0.1%
(wt/vol) SDS, and Mini EDTA-free protease inhibitors (Roche Molecular Biochemicals, Laval, Québec, Canada)], from
both treated and untreated (control) cells, were normalized for protein
concentration, using a DC protein assay (Bio-Rad Laboratories, Inc.). Equal amounts of total protein per sample were dissolved
in nondenaturing SDS sample buffer [0.5 M Tris (pH 6.8),
1% SDS, 10% glycerol, and bromphenol blue] and boiled for 5 min.
Samples were electrophoresed on 15% SDS-polyacrylamide gels, then
electroblotted onto nitrocellulose, and membranes blocked with 4%
milk-TBS-T [Tris-buffered saline-Tween-20 (0.1%)] for 1 h at 22
C. Western blots were incubated with IGFBP-rP1, -rP2, or -rP3 antisera
at a 1:3000 dilution and with p16INK4a IgG
antibody at a dilution of 1:500 (1 µg/ml) in TBS-T overnight at 4 C.
Blots were washed with TBS-T and then incubated for 1 h at 22 C
with a 1:3000 dilution of horseradish peroxidase-linked antirabbit or
antimouse IgG secondary antibodies. Proteins of interest were detected
with ECL chemiluminescence reagents, according to the manufacturer’s
protocol.
RNA analyses
Total cytoplasmic RNA was isolated from cells, by use of RNeasy
(QIAGEN, Inc., Chatsworth, CA). Twenty micrograms of each
RNA preparation were electrophoresed on a 1.2% agarose-2.2
M formaldehyde gel, transferred overnight onto a nylon
membrane (GeneScreen), using 10 x SSC as the transfer solution,
and cross-linked to the membrane by UV irradiation in a Stratalinker
1800 (Stratagene). The Northern blots were then probed
with an EcoRI/Xho fragment of IGFBP-rP1 (27), or a
BamHI/Xho fragment of IGFBP-rP2 (28), which were
radiolabeled (1 x 109 dpm/µg) with
[
-32P]deoxycycidine triphosphate (NEN Life Science Products-DuPont; SA, 3000 Ci/mmol) using a random
priming kit (Prime-a-Gene, Promega Corp.). Northern blots
were hybridized overnight at 65 C in hybridization buffer (Rapid-Hyb,
Amersham Pharmacia Biotech), according to the
manufacturer’s instructions. Blots were then washed for 15 min in
2 x SSC/0.1% SDS at 22 C, followed by two more stringent washes
in 0.2 x SSC/0.1% SDS at 65 C for 15 min. Blots were exposed to
Kodak Biomax film (Eastman Kodak Co.,
Rochester, NY) for 12 to 48 h at -70 C, using one intensifying
screen. Membranes were then reprobed with 18S ribosomal RNA, which
acted as a loading control for the RNA samples. An image analyzer
(GS-700) equipped with MultiAnalyst version 1.0.2 Software
(Bio-Rad Laboratories, Inc.) was used to quantify the
resulting bands.
RT-PCR
RT-PCR was performed using
5'-CGCGAATTCGCCATGCAGAGTGTGCAGAGCACG-3' and
5'-GGGGCTCGAGTTACATTTTCCCTCTGGTAGTC-3' primers specific for IGFBP-rP3.
One microgram of total RNA from each cell line was reversed transcribed
in a vol of 20 µl, by use of Reverse Transcription System Kit
(Promega Corp.), following the manufacturer’s
instructions. The reaction was performed at 42 C for 15 min, denatured
at 99 C for 5 min, and placed on ice. One microliter of the mixture and
50 pmol of 5' and 3' primers were employed in PCR amplification
reactions using Advantage GC cDNA PCR Kit (CLONTECH Laboratories, Inc., Palo Alto, CA). Amplification of the cDNA was carried out
with 25 cycles of denaturing at 94 C for 1 min, annealing at 55 C for 1
min, and extension at 72 C for 2 min. One negative and one positive
control were included in all reactions.
Statistical analyses
All experiments were performed at least twice. Statistical
analyses were performed by a two-tail Student’s t test,
assuming unequal variances, using Excel Data Analysis Software
(Microsoft Corp., Redmond, WA). Data are expressed as
means ± SE. P < 0.05 was
considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In the malignant
LNCaP, DU145, and PC-3 cells, IGFBP-rP1 mRNA was undetectable by
Northern blot, and an immunoreactive band was only present in cell
lysates but not in conditioned media from these cells.
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).
IGFBP-rP3 message was evaluated by RT-PCR in only a limited number of
cell lines but was identifiable in P69, M12, LNCaP, and PC-3 cells.
IGFBP-rP3 protein was undetectable in conditioned media from HPEC,
compared with readily detectable levels in the immortalized P69 cell
line and malignant M12, DU145, and PC-3 cells (Fig. 2).
Expression of IGFBP-rP1 and 2 during senescence of HPEC
HPEC cells have a limited life span, with no more than 30
population doublings before they enter replicative senescence
(corresponding to our 9th culture passage). Thus, early passages of
HPEC (<4th) are highly replicative cells (duplication time for 4th
passage is approximately 2 days), whereas senescent cells (>9th
passage) are unable to replicate, and they die over time (Fig. 3).
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, A and B).
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).
IGFBP-rP2 is responsive to growth regulators in HPECs
Because TGF-ß, atRA, and IGF-I are important regulators of
prostate epithelial growth and survival (32, 33, 34) and
because IGFBP-rP2 is tightly regulated by TGF-ß in other cellular
systems, we wished to investigate the effects of these growth
inhibitors (TGF-ß and atRA) and growth stimulator (IGF-I) on cell
proliferation and IGFBP-rP2 expression in our normal and malignant
prostate cells.
Both TGF-ß and atRA caused a dose-dependent inhibition of cellular
growth in the normal HPEC (early replicative passages) cultured for
48 h under serum-free conditions, whereas only TGF-ß was
inhibitory for the malignant PC-3 cells (Fig. 5). Paralleling the inhibition of
proliferation of HPEC by TGF-ß and atRA, there was a dose-dependent
increase in the concentrations of both IGFBP-rP2 protein and
steady-state mRNA. A maximum protein response equivalent to a 6-fold
increase over the nontreated cells was observed at a dose of TGF-ß1
of 5 ng/ml, whereas 1 µM atRA caused an increase of
IGFBP-rP2 protein of about 4-fold over basal levels (Fig. 6A). Similar changes were also observed
in the concentrations of IGFBP-rP2 steady-state mRNA (Fig.
6B).
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, A and B).
The effects of IGF-I were also tested in the normal HPEC (late,
presenescent passages), where it exerted clear mitogenic actions (Fig. 5A). Importantly, IGFBP-rP2 was also regulated by IGF-I in HPEC, both
at the protein and at the mRNA level, with a 90% reduction of both
secreted protein and steady-state mRNA, upon treatment with IGF-I at a
dose of 100 ng/ml (Fig. 6, C and D). In contrast to IGFBP-rP2, neither
IGFBP-rP1 protein nor its mRNA experienced significant changes in
response to IGF-I treatment in HPEC (Figs. 6, C and D).
In PC-3 cells, only the effects of TGF-ß and atRA on IGFBP-rP2
concentrations were studied. TGF-ß produced a significant increase
(4-fold) in protein levels over nonstimulated values. Similarly,
treatment with atRA, under the same experimental conditions, resulted
in a 6-fold increase in IGFBP-rP2 concentrations (Fig. 7A). These changes did not, however,
follow parallel increases in the concentrations of steady-state mRNA,
as the levels of IGFBP-rP2 mRNA did not significantly change on TGF-ß
treatment and were only increased 2-fold by atRA (Fig. 7B).
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IGFBP-rP3 is regulated in HPECs
IGFBP-rP3 has been previously shown to be expressed in prostate
epithelial cells, but no regulation has been described, as yet, in
these cultures (22). To better understand the role of this
protein in the human prostate, we also investigated the effects of
TGF-ß, atRA, and IGF-I on the expression of IGFBP-rP3 in
vitro.
IGFBP-rP3 was not produced by normal prostate epithelial cells, nor was it induced by any of the above-mentioned growth regulators in these cells. In the well-established PC-3 (and in DU145) cancer cells, IGFBP-rP3 was readily detected in conditioned media under serum-free incubation, but no significant regulation was observed in these cultures.
In the P69/M12 lineage, both TGF-ß and atRA also produced a
dose-dependent inhibitory effect on cellular proliferation (Fig. 8). Interestingly, atRA, but not TGF-ß,
down-regulated the concentrations of secreted IGFBP-rP3 in both P69 and
M12 cells (4- and 2-fold, respectively; Fig. 9).
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) but had
no observable effects on the concentrations of secreted IGFBP-rP3
protein (Fig. 9). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IGFBP-rP1 has been shown to be differentially expressed in normal meningeal, breast, and prostate cells, compared with their malignant counterparts (11, 16, 35). In the prostate, it was suggested that IGFBP-rP1 might have an antiproliferative effect, because it was also up-regulated by the epithelial cell growth inhibitors TGF-ß1 and atRA (16). Indeed, a more recent study indicates a possible role of IGFBP-rP1 as a tumor suppressor gene for prostate cancer, because overexpression of this gene in the tumorigenic and metastatic M12 cells caused an antiproliferative effect in vitro and in vivo (36). More difficult to reconcile, however, is a recent report indicating an up-regulation of IGFBP-rP1 during prostate carcinogenesis (37). Because the results by Degeorges et al. are based on immunohistochemistry studies, it is possible that the differences found are attributable to alternative properties or specificity of their antibody; alternatively, different sources of prostatic epithelial cells may vary in these properties.
Further evidence supporting the role of IGFBP-rP1 as a tumor suppressor gene relates to its up-regulation during senescence of normal epithelial cells. Swisshelm et al. (35) have described an enhanced expression of IGFBP-rP1 in senescent human mammary epithelial cells, indicating a possible involvement of this protein in the cell-cycle mechanisms leading to cellular senescence.
HPEC, similarly to human mammary epithelial cells, have a limited life span, undergoing senescence after a limited number of population doublings. Besides a failure to replicate in vitro, senescent HPECs exhibit phenotypic changes, as they become larger and flattened, and induce ß-galactosidase activity (data not shown). Consistent with this, a marked up-regulation of the cell-cycle inhibitor p16INK4a was observed at late passages of HPEC, a phenomenon that has been recently reported in these cells (31). Our results are, thus, in agreement with observations in human mammary cells, in that the concentrations of both IGFBP-rP1 mRNA and protein were markedly enhanced during replicative senescence of normal prostate epithelial cells. Thus, although the precise role of IGFBP-rP1 in the human prostate has, as yet, to be clarified, our results support the hypothesis that IGFBP-rP1 is a tumor suppressor gene and/or senescence factor.
Several lines of investigation also support roles of IGFBP-rP2 in growth regulation and tumorigenesis. IGFBP-rP2 mediates most of the biological actions of TGF-ß in fibroblasts where a novel TGF-ß response element in the IGFBP-rP2 promoter has been found (19). Additionally, IGFBP-rP2 is a growth factor for endothelial cells (29, 38). Supporting its role in tumorigenesis, IGFBP-rP2 was expressed by a chondrosarcoma-derived chondrocytic cell line and a fibrosarcoma cell line in vitro (29, 39) and has been found in the stromal component of breast and pancreatic cancer and desmoplastic melanomas (40, 41, 42). Additionally, it has been shown that IGFBP-rP2 is up-regulated by TGF-ß1 in the breast cancer cell line Hs578T (25) and that IGFBP-rP2 induces apoptosis in the estrogen receptor-positive breast cancer cell line MCF-7 (43). These lines of evidence suggest that IGFBP-rP2 plays also a role in modifying the growth of stroma in desmoplastic tumors and in regulating the growth of breast cancer cells.
In this light, our results indicate that IGFBP-rP2 may also regulate the growth of normal and prostate cancer cells, because it is noticeably up-regulated by growth inhibitory factors, such as TGF-ß and atRA, in normal and malignant cells, and down-regulated by growth-promoting factors, such as IGF-I, in HPEC. In the prostate, TGF-ß is known to limit the proliferation and survival of the normal epithelium, an effect that is lost in malignant cells largely because of a down-regulation of TGF-ß receptors during prostate carcinogenesis (44). Retinoids also exert potent inhibitory properties in the normal prostate and have been shown to be present at lower concentration in prostate cancer tissues (33, 45). Thus, both TGF-ß and atRA play major roles in controlling the growth and survival of normal prostate cells and probably also in the development of prostate cancer. IGFs regulate also the proliferation of prostate cells (34). Interestingly, the expression of type I IGF-I receptor, which mediates most of the biological activities of IGFs, in markedly down-regulated during carcinogenesis (3). Thus, IGFs seem to play fundamental roles in the regulation of growth and malignant transformation of normal prostate epithelial cells. Because the above-mentioned growth regulators affected the concentrations of both IGFBP-rP2 protein and steady-state mRNA in our cultured cells, we speculate that, in the human prostate, IGFBP-rP2 is a downstream effector of growth inhibitors and that IGFBP-rP2 expression must be down-regulated by growth factors to support cell proliferation.
Further observations support the hypothesis that IGFBP-rP2 may act as a growth inhibitor. A striking increase of IGFBP-rP2 was observed during senescence of HPECs, paralleling increases of both IGFBP-rP1 and the cell cycle inhibitor p16INK4a. Thus, IGFBP-rP2 could also be a downstream effector in the mechanisms leading to cellular senescence of normal cells. In summary, IGFBP-rP2 is a potential growth inhibitor that is induced during senescence in prostate epithelial cells and can mediate the effects of growth inhibitors on cell-cycle progression and/or apoptosis in normal and malignant prostate.
IGFBP-rP3 is overexpressed in Wilms tumor, showing an inverse correlation with the concentrations of the tumor-suppressor gene WT1(21), which suggests a potential role of this protein as an protooncogene. Supporting this hypothesis is the observation that overexpression of the aminoterminal truncated NOV molecule was also able to transform chicken embryo fibroblasts (20). More recently, NOV has also shown to induce proliferation of mouse fibroblasts in vitro and to enhance phosphorylation of a 221-kDa protein, suggesting growth-stimulatory properties of this protein through activation of a still unidentified phosphorylated molecule (46).
In the prostate, data on NOV expression are only modest, with one report showing, by RT-PCR, that NOV mRNA is preferentially expressed in PC-3, compared with other normal and malignant prostatic epithelial cells (22). Our results are consistent with this report, and we have extended these findings by demonstrating that IGFBP-rP3 protein was detectable in the condition medium from PC-3 cells. Moreover, by RT-PCR, we were able to detect IGFBP-rP3 mRNA in the immortalized P69 cells and in the malignant M12 and LNCaP cell lines. IGFBP-rP3 protein was also detected in conditioned media from these cells (except LNCaP cells) and in the conditioned medium from the malignant DU145 cells. However, IGFBP-rP3 expression was not detected in normal HPEC cells. Additionally, IGFBP-rP3 expression was suppressed by the growth-inhibitor atRA in P69 and M12 cells. Thus, IGFBP-rP3 expression in prostatic cells is consistent with its potential role as a protooncogene and/or growth factor.
In summary, we report on the expression and regulation of two additional IGFBP-rPs in prostate cells, IGFBP-rP2 and rP3, with results that support the hypothesis that these proteins, like IGFBP-rP1, are involved in the regulation of prostatic cell growth. IGFBP-rP2 may play a role as a growth inhibitor, because its expression is: 1) enhanced during senescence of normal prostate epithelial cells (in a fashion similar to that of IGFBP-rP1); 2) increased by growth inhibitory factors (TGF-ß and atRA); and 3) decreased by IGF-I. Conversely, IGFBP-rP3 may act as a growth stimulator for prostate cells, given its preferential expression in malignant cells and its down-regulation by atRA.
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Footnotes |
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2 Supported by a Fellow Research Funding Grant from Eli Lilly & Co. Also, a recipient of Grants 97/5309 and 98/9198 from the
Fondo de Investigación Sanitaria, Spain.
Received April 5, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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6-fold) of p16INK4a concentrations
at late, low-replicative passages, compared with early ones, as part of
the senescence process of HPEC cells.
