TRANSFORMING growth factor- (TGF ) is a pleiotropic

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1 /99/$03.00/0 Vol. 140, No. 8 Endocrinology Printed in U.S.A. Copyright 1999 by The Endocrine Society Transforming Growth Factor- 1-Induced Proliferation of the Prostate Cancer Cell Line, TSU-Pr1: The Role of Platelet-Derived Growth Factor* SHARON M. SINTICH, MARILYN L. G. LAMM, JULIA A. SENSIBAR, AND CHUNG LEE Department of Urology, Northwestern University Medical School, Chicago, Illinois TRANSFORMING growth factor- (TGF ) is a pleiotropic growth factor that regulates cell growth, differentiation, extracellular matrix production, cell motility, angiogenesis, and immunosuppression. As a general rule, it acts as a growth inhibitor for cells of epithelial origin, whereas it acts as a growth stimulatory factor for mesenchymal cells (1). For most prostate cancer cells, TGF is known to inhibit cell proliferation (2). Unexpectedly, the results of a recent study demonstrated a proliferative effect of TGF 1 on a human prostate cancer cell line, TSU-Pr1 (3). This cell line is androgen independent and was derived from a lymph node metastasis that developed in a 73-yr-old male with moderately differentiated cancer (4). Currently, there is no evidence to indicate that TGF can directly stimulate cellular proliferation, although it can act indirectly by up-regulating the expression of mitogenic factors (5). Platelet-derived growth factor (PDGF) is a homo/heterodimeric growth factor that is composed of an A and/or a B chain. There are three active forms, denoted PDGF-AA, -BB, and -AB. These growth factors can stimulate cellular proliferation, differentiation, migration, and angiogenesis (6). TGF has been shown to modulate the expression of either the PDGF-A or -B chain in different cell types (7 9). In this study, we explore the possibility that TGF 1 may mediate a proliferative effect on TSU-Pr1 cells through the PDGF signaling system. We demonstrate that PDGF expression is up-regulated after treatment with TGF 1 and that PDGF stimulates proliferation. Therefore, PDGF plays a role in TGF 1-induced proliferation in TSU-Pr1 prostate cancer cells. Received August 31, Address all correspondence and requests for reprints to: Dr. Chung Lee, Department of Urology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois * This work was supported in part by NIH Grants CA and CA ABSTRACT The results of our previous study revealed that transforming growth factor- 1 (TGF 1) stimulated proliferation of the prostate cancer cell line, TSU-Pr1. This observation is unexpected, for TGF usually inhibits proliferation in prostate cancer cells. The present study examines possible mechanisms through which TGF 1 induces this proliferation. We postulate that TGF 1 action is mediated through an indirect mechanism by inducing the expression of plateletderived growth factor (PDGF), which, in turn, stimulates proliferation. The TGF 1-induced proliferation can be abrogated by treatment with a PDGF-neutralizing antibody. Treatment with exogenous PDGF significantly increased TSU-Pr1 proliferation. Finally, treatment of TSU-Pr1 cells with TGF 1 resulted in an increase in PDGF secretion. These results indicate that TGF 1-induced proliferation in TSU-Pr1 cells is at least mediated through an increased secretion of PDGF. (Endocrinology 140: , 1999) Cell culture Materials and Methods TSU-Pr1 cells were provided by Dr. Joel Nelson (Johns Hopkins University, Baltimore, MD), and PC3 cells were obtained from American Type Culture Collection (Manassas, VA). These cells were routinely maintained in medium [RPMI 1640 containing penicillin (100 U/ml)/ streptomycin (100 g/ml); Life Technologies, Inc., Gaithersburg, MD] supplemented with 10% FBS (Summit Biotechnology, Fort Collins, CO) ina37c,5%co 2 incubator. For the following experiments, these cells were cultured in serum-free RPMI 1640 rather than serum-containing medium. [ 3 H]Thymidine Incorporation TSU-Pr1 cells were seeded at approximately cells/well (24- well plate) and were incubated for 22 h in 1 ml serum-free culture medium containing TGF 1 (R&D Systems, Minneapolis, MN) at preselected concentrations. One microcurie per well of [ 3 H]thymidine (6.7 Ci/mm; Amersham, Arlington Heights, IL) was added to the culture, and incubation was continued for an additional 4 h. Radioactivity incorporated into the cells was trichloroacetic acid precipitated and counted with a scintillation counter as counts per min. Determination of total cell number Cellular proliferation was assayed by total cell counts. Cells were seeded at approximately cells/well (24-well plate) and allowed to adhere overnight. The cells were then treated with the appropriate growth factors and/or antibodies. After 48 h, the cells were detached with 0.5 ml 0.35% trypsin-0.1% EDTA solution. The cell solution was transferred to a counting vessel containing 9.5 ml isotonic solution and counted using a Coulter counter (Coulter Corp., Hialeah, FL). Results were expressed as a percentage of the cells in the control culture. RNA isolation and RT-PCR Isolation of messenger RNA (mrna) was performed according to the manufacturer s recommended protocol for the Quickprep Micro mrna purification kit (Pharmacia Biotech, Uppsala, Sweden). RT-PCR for PDGF receptor- and - was performed using the Gene Amp RNA kit (Perkin-Elmer, Norwalk, CT) as previously described (10, 11). For receptor-, the 5 -primer was 5 -CTGGAAGAAATCAAAGTCCCATCC- 3, and the 3 -primer was 5 -TGAGCCATCCTGTGATCATCGAACC

2 3412 PDGF IN TGF 1-INDUCED PROLIFERATION IN TSU-PR1 CELLS Endo 1999 Vol 140 No 8 For receptor-, the 5 -primer was 5 -GACCACCCAGCCATCCTTC-3, and the 3 -primer was 5 -GAGGAGGTGTTGACTTCATTC-3. The PCR reaction was run for 35 cycles of 1 min at 95 C, 2 min at 65 or 58 C, and 2 min at 72 C. The PCR products were visualized on a 1% agarose gel and verified by restriction digestion with BglI and StyI accordingly. Growth factors and antibodies For experiments using the neutralizing antibody, TSU-Pr1 cells were treated with 10 ng/ml TGF 1 in the presence and absence of neutralizing antibodies to PDGF-AA and PDGF-BB (0.1, 1.0, and 10 g/ml). As a control, the cells were also treated with TGF 1 plus goat IgG protein (Sigma Chemical Co., St. Louis, MO) at the same concentrations. For the PDGF response assay, the cells were treated with various concentrations of PDGF-AA and PDGF-BB (0.001, 0.01, 0.1, 1.0, and 10 ng/ml). All growth factors and antibodies were obtained from R&D Systems. Enzyme-linked immunosorbent assay (ELISA) for PDGF-AB TSU-Pr1 cells were treated with 10 ng/ml TGF 1 for various time periods (3, 6, 12, and 24 h). The conditioned medium was collected and centrifuged to clear the medium of floating cells. The media was tested with an ELISA (R&D Systems) according to the manufacturer s instructions. Trypan blue dye exclusion assay PC3 and TSU-Pr1 cell viability was assessed by treating the cells with various concentrations (1 and 10 ng/ml) of TGF 1 for 24 h as described above. After 24 h, the cells were detached with trypsin and pelleted by centrifugation. Trypan blue dye (Sigma Chemical Co.) was added to each pellet, and the number of cells that absorbed the dye was visually counted. Results Effect of TGF 1 on proliferation and cell death Treatment with TGF 1 stimulated proliferation in the prostate cancer cell line TSU-Pr1. Figure 1a shows a dosedependent increase in [ 3 H]thymidine incorporation in the presence of TGF 1. The mean increases in [ 3 H]thymidine incorporation were 99%, 225%, and 272% after the addition of 0.1, 1.0, and 10 ng/ml TGF 1, respectively. Experiments measuring total cell number in response to TGF 1 treatment exhibited the same result (Fig. 1b). As TGF 1 treatment is also known to induce apoptosis (12), trypan blue exclusion was used as a simple method to assess the extent of cell death induced by TGF 1. As Table 1 indicates, TGF 1 significantly increased the extent of cell death in PC3 cells (20%), whereas this treatment did not affect TSU-Pr1 cell death. These results suggest that TSU-Pr1 cells also show an unusual response to TGF -induced apoptosis. Effect of PDGF on proliferation As TGF 1 is not known to be a direct cellular mitogen, we hypothesized that TGF 1 treatment would induce the expression of a proliferative factor in an autocrine manner. PDGF was considered a candidate for the mitogenic growth factor, because its expression is up-regulated by TGF in mesenchymal cells and in some cancer cells (6, 13). In addition, TSU-Pr1 cells expressed the message for the PDGF receptor type. Figure 2 demonstrates the expression of the -form by RT-PCR with prostate stroma as a positive control. PDGF receptor was not expressed by TSU-Pr1, but was detected in the positive control. Total cell number was measured after treatment with either PDGF-AA or PDGF-BB. FIG. 1. a, Effect of TGF 1 on DNA synthesis in TSU-Pr1. Cells were incubated in varying concentrations of TGF 1. At 22 h of culture, [ 3 H]thymidine was added for 4 h more. The radioactivity incorporated into the cells was counted and expressed as counts per min/well. Data are presented as a percentage of the untreated control value, and each bar represents the SD of three or four replicates per experiment. Results are representative of three experiments. DNA synthesis was significantly increased (*, P 0.01; **, P 0.001). b, Effect of TGF1 on cell number in TSU-Pr1. Cells were incubated in varying concentrations of TGF1. After 48 h of culture, cell number was determined with a Coulter counter. Data are presented as a percentage of the untreated control value and are representative of three experiments, with four replicates per experiment. The vertical bar represents the SD. *,P Results were expressed as the percent increase in total cell number compared with that in the control culture. Figure 3 reveals a dose-related increase in cell number of 28% with PDGF-AA and 23% with PDGF-BB. Effect of TGF 1 on PDGF-mediated proliferation To determine whether PDGF mediates TGF 1-induced proliferation in TSU-Pr1 cells, a neutralizing antibody to

3 PDGF IN TGF 1-INDUCED PROLIFERATION IN TSU-PR1 CELLS 3413 TABLE 1. Effect of TGF 1 on cell viability % of viable cells Cell line 0 ng/ml TGF- 1 1 ng/ml TGF 1 10 ng/ml TGF 1 PC a a TSU-Pr Cell viability was assessed by the trypan blue exclusion test. Each value represents the mean SD of two separate experiments. a The value is significantly decreased compared to that in the untreated (0 ng/ml TGF- 1) group (P 0.05). FIG. 3. Effect of PDGF on TSU-Pr1 proliferation. Cells were treated with increasing concentrations of PDGF-AA ( ) and PDGF-BB (f). After 48 h, the total cell number was determined as described in Materials and Methods. The cell number was significantly increased (*, P 0.05; **, P 0.005) with all treatments. Results are representative of three experiments. Each bar represents the SD. FIG. 2. Detection of PDGF receptor expression in TSU-Pr1 cells by RT-PCR. Cells were grown under normal conditions, and mrna was isolated as described in Materials and Methods. A 1% agarose gel is shown in which A represents a 100-bp ladder, C designates a positive control of prostate stromal cells, and T is TSU-Pr1 cells., Expression of PDGF receptor- ;, PDGF receptor- expression. The PCR products were the expected sizes of 501 bp for and 228 bp for. either PDGF-AA or PDGF-BB was used. Total cell number was determined after treatment with TGF 1 in the presence and absence of PDGF antibodies. Again, results were expressed as the percent increase in total cell number compared with that in the control culture. Figure 4 shows that at 10 ng/ml, TGF 1 significantly increased the total cell number (58%). The addition of neutralizing antibodies to both PDGF-AA and PDGF-BB (1.0 and 10 g/ml) abrogated the stimulatory effect of TGF 1 and caused cell number to remain at basal levels, similar to those in the untreated controls. This elimination of the TGF 1 effect was specific for the PDGF antibodies, as treatment with goat IgG protein had no effect on the stimulation. Effect of TGF 1 on PDGF secretion It is important to demonstrate that TGF 1 treatment leads to an increase in PDGF secretion. TSU-Pr1 cells were treated with 10 ng/ml TGF 1, and the conditioned medium was collected at various time points (3, 6, 12, and 24 h). An ELISA for PDGF-AB was performed on the collected conditioned medium. Figure 5 indicates that treatment with TGF 1 significantly increased the secretion of PDGF, reaching a maximum at 3 h. The levels decreased at 6 h, but remained significantly elevated for the duration of the experiment. FIG. 4. Effect of neutralization of PDGF on TGF 1-induced proliferation in TSU-Pr1 cells. Cells were treated with 10 ng/ml TGF 1 for 48 h in the presence (0.1, 1.0, or 10 ng/ml) or absence of PDGF-AAand PDGF-BB-neutralizing antibodies or isotype control antibodies (goat IgG). The TGF -induced proliferation was significantly decreased (*, P 0.05) by PDGF antibodies. Data are presented as a percentage of the untreated control values in three experiments. Each bar represents the SD. These results are consistent with the concept that PDGF plays a role in TGF 1-induced proliferation in TSU-Pr1 cells. Discussion The human prostate cancer cell line, TSU-Pr1, unlike other prostate cancer cells, undergoes proliferation in response to TGF 1. The results of the present study have demonstrated that the proliferative effect of TGF 1 is mediated through an autocrine induction of PDGF. The supportive evidence came from the observation that neutralizing antibodies to both

4 3414 PDGF IN TGF 1-INDUCED PROLIFERATION IN TSU-PR1 CELLS Endo 1999 Vol 140 No 8 FIG. 5. Effect of TGF 1 on PDGF secretion by TSU-Pr1 cells. Cells were treated with 10 ng/ml TGF 1 for different periods of time (3, 6, 12, and 24 h). At each time point the conditioned medium was assayed with an ELISA for PDGF-AB. The level of PDGF-AB was significantly increased (*, P 0.05) after 3hinthepresence of TGF 1. Data are presented as picograms per 10,000 cells. Each point represents the mean SD of duplicate wells. Results are representative of two experiments. PDGF-A chain and PDGF-B chain can abrogate the stimulatory effect of TGF 1 treatment. In addition, PDGF-AB secretion into TSU-Pr1-conditioned medium is increased in the presence of TGF 1. In mesenchymal cell systems, both PDGF-A chain and PDGF-B chain have been shown to be up-regulated in response to TGF (14 16). These findings are in agreement with the few cases of TGF -induced proliferation in epithelial cells where PDGF expression has been shown to be increased after TGF treatment (13, 17). For example, in the U9 subline of the colon carcinoma cell line HT29, TGF induces proliferation and increases PDGF expression, which enhances angiogenesis (13). On the other hand, our findings are novel in that this is the first incidence where the primary effect of TGF -induced PDGF expression resulted in increased cellular proliferation and not indirect tumor effects such as migration or angiogenesis. The proliferative increase in response to exogenous recombinant PDGF was modest compared with the strong stimulation detected after TGF 1 treatment. Our ELISA data suggest that TGF 1 sustains an increased production of TGF 1; therefore, a possible reason for the lack of comparable responses of proliferation shown in Figs. 1 and 3 may be related to the half-life of the exogenously added PDGF. In general, mesenchymal cells, unlike epithelial cells, express PDGF receptors, which allows PDGF to elicit a growth response in these cells (18). PDGF receptors have been shown to be up-regulated by TGF in fibroblast cells such as 3T3 mouse fibroblasts (19), human skin fibroblasts (20), and foreskin fibroblasts (6). In the present study, the observation of a mitogenic response to PDGF by TSU-Pr1 cells implies that these cells contain functional PDGF receptors. An important requisite for TSU-Pr1 cells to undergo TGF -induced proliferation is that these cells should not only be sensitive to the proliferative effect of PDGF, but they should also respond to TGF by secreting increasing levels of PDGF. The present results have demonstrated that TSU-Pr1 cells have met this requirement. This may be one of the reasons why TGF stimulates growth in these cells but not in other prostate cancer cells. Other prostate cancer cell lines, such as PC3 and DU145, are also growth stimulated by PDGF, but are strongly growth inhibited by TGF (2, 21). This phenomenon indicates that within prostate cancer cells there may be differences in signaling that allow TSU-Pr1 cells to be insensitive to the growth inhibitory effects of TGF. The results of our earlier study have demonstrated that TSU-Pr1 cells have functional TGF receptors (3). However, it remains possible that one of the downstream events of TGF signaling, which leads to growth arrest, may be defective in TSU-Pr1 cells. It has been hypothesized that the numerous responses to TGF are mediated by different signaling pathways (4). It is possible that the pathway that invokes the induction of PDGF is independent of that which induces apoptosis or growth arrest. Our data presented in Table 1 reveal that TGF 1 did not have an effect on TSU-Pr1 cell viability. This indicates that the machinery for TGF -induced apoptosis may be altered in TSU-Pr1 cells, which allows these cells to escape the inhibitory effects of TGF. (12). Furthermore, as TGF 1 can up-regulate cyclin-dependent kinase inhibitors such as p15, p21, and p27, there may be a defect in these cell cycle regulators in TSU-Pr1 cells that disables the TGF growth arrest machinery (22). These aspects of TGF response in TSU-Pr1 cells are the topics of future investigations. The TSU-Pr1 cell line is a highly aggressive, androgenindependent cell line that represents late stage prostate cancer (4). It is possible that the proposed model of TGF upregulation of PDGF described in this study may operate in vivo. PDGF receptor- and - have been detected in both normal and cancerous prostate tissue (23, 24). TSU-Pr1 cells only express the PDGF receptor-. In addition, the TGF signaling system has been extensively studied in prostate tumors. It is known that prostate cancer specimens express both TGF and its receptors (25). The results of our earlier studies have provided a critical piece of information that supports a stimulatory role for TGF in prostate cancer. In a recent study, we noted that TSU-Pr1 cells express a large amount of clusterin. If the effective level of clusterin is reduced either by the use of a specific antibody or by treatment with antisense oligonucleotides to clusterin, TSU-Pr1 cells will undergo apoptosis in response to TGF 1 (Sintich, S. M., L. Januis, T. Yang, J. A. Sensibar, and C. Lee, manuscript submitted). We also reported that clusterin overproduction correlates with advanced stages of prostate cancer (26). It is likely that in a clinical setting, overexpression of clusterin protects cancer cells from the antiapoptotic effect of TGF, which, in turn, induces the expression of PDGF. These studies indicate that the requirements for TGF -stimulated proliferation are present in prostate tumors. In summary, the present results demonstrate an important role for PDGF in TGF 1-induced proliferation in TSU-Pr1 cells. Proliferation in these cells is also stimulated by both PDGF-AA and -BB, whereas neutralization of either of these factors abrogates the stimulatory effect of TGF 1. Furthermore, treatment with TGF 1 increases the secretion of PDGF-AB into TSU-Pr1-conditioned medium. These data

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