This observation confirmed our preliminary results (12). Open in a separate window Fig. at high concentrations of TGF-1 (1.0 and 10 ng/ml). An inhibitor of cyclin-dependent kinase (cdk), p15INK4b, was up-regulated at both transcript and protein levels in these cultures by TGF-1 in a dose-related manner as determined by RT-PCR and Western blot analysis. The transcript, but not the protein, for another cdk inhibitor, p21Cip1, was up-regulated with treatment of TGF-1 to these cells. Levels of other cdk inhibitors, such as p16INK4a and p27Kip1, were constitutively expressed in prostatic stromal cells and were not significantly affected by TGF-1 treatment. Finally, the growth arrest effect of TGF-1 was abrogated when antisense oligonucleotides to p15INH4b, but not p21Cip1, were added to the culture medium. These data indicate that the dual effect of TGF-1 is mediated, at least, by up-regulation of PDGF-BB and p15INK4b, respectively. Abbreviations: BPH, Benign prostatic hyperplasia; cdk, cyclin-dependent kinase; FBS, fetal bovine serum; PDGF, platelet-derived growth factor; Smad, signaling molecules identified downstream of TGF superfamily ligands TGF- IS THE prototypic member Aminoacyl tRNA synthetase-IN-1 of a superfamily. TGF-1, -2, and -3 have been identified in mammals (1) and they are able to mediate a wide range of cellular events (2C4). In prostatic stromal cells, all three types of mammalian TGF- isoforms are expressed (5), with TGF-1 being the predominant one (6). Cellular responses of prostatic stromal cells to TGF- have been inconsistent. In most studies, TGF- has been shown to inhibit proliferation in prostatic stromal cells (7C9). But in few reports, it has a stimulatory effect (10, 11). The above discrepancy has been resolved in our preliminary studies (12) in which TGF- showed both stimulatory and inhibitory effects on growth of prostatic stromal cells, depending on the concentrations used in the cultures. At low concentrations TGF-1 induced proliferation, whereas at high concentrations, it induced growth arrest. In other Aminoacyl tRNA synthetase-IN-1 cell systems, the inhibitory effect of TGF- has been linked to the induction of inhibitors to cyclin-dependent protein kinases (cdk) (13, 14). The stimulatory effect of TGF- has been linked to the expression of platelet-derived growth factor (PDGF) (15). In the present study, we explored the possible mechanisms of these seemingly conflicting events mediated by TGF- in human prostatic stromal cells. Materials and Methods Cell culture Prostatic stromal cells were derived from 10 surgical specimens from men who were subjected to surgery for the treatment of bladder neck obstruction secondary to benign prostatic hyperplasia (BPH). The use of human surgical specimens was approved by the institutional review board, with the understanding that the identity of patients would not be revealed. Primary cultures of prostatic Mouse monoclonal to ERBB3 stromal cells were established according to the methods reported earlier (16, 17). Briefly, freshly isolated tissue specimens were mechanically and enzymatically dissociated by treatment with Dnase (Sigma, St. Louis, MO) and collagenase (Sigma). Epithelial cells were separated from stromal cells by discontinuous Percoll (Sigma) gradient centrifugation. Stromal cells were then cultured in phenol red-free RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). Cells used in this study were derived from 2nd to 15th passages. [3H]Thymidine incorporation Prostatic stromal cells were plated in 24-well plates at a concentration of 2 104 cells/ml in phenol red-free RPMI 1640 medium supplemented with 10% FBS. The medium was changed to serum free RPMI 1640 with 1% ITS (insulin, transferrin, seleneous acid; Aminoacyl tRNA synthetase-IN-1 BD Biosciences, Bedford, MA) for 24 h. To test the effect of TGF-1 on proliferation, the medium was changed to a 1% ITS or 1% ITS supplemented with various concentrations of TGF-1 (R&D Systems, Minneapolis, MN) at 0.001, 0.01, 0.1, 1.0, or 10 ng/ml, with medium change every other day. After 5 d of incubation, [3H]thymidine (Amersham Pharmacia, Piscataway, NJ) was added at a dosage of 1 1.0 Ci/well for 24 h. DNA was precipitated using 10% trichloroacetic acid at 4 C for 20 min, Aminoacyl tRNA synthetase-IN-1 followed by 0.4 n NaOH at 37 C overnight. [3H]Thymidine incorporation into cellular DNA was measured with a scintillation counter and was expressed as counts per minute. To determine the effect of autocrine production of PDGF (R&D Systems), cells were incubated in medium containing 0.001 ng/ml TGF-1 with neutralizing antibody (0.5 g/ml) against PDGF-AA or -BB to determine their effects on growth of prostatic stromal cells in culture. [3H]Thymidine incorporation assay was carried out in these cells as well. PDGF-BB quantitation Prostatic stromal cells were seeded at 2 .
This observation confirmed our preliminary results (12)