Transforming growth factor-beta 1 is a mediator of androgen-regulated growth arrest in an androgen-responsive prostatic cancer cell line, LNCaP

LNCaP is an androgen-responsive prostatic cancer cell line that exhibits a bell-shaped growth response to increasing doses of dihydrotestosterone (DHT) in culture. Although the precise mechanism responsible for this growth response to androgen stimulation remains unclear, many studies have suggested that androgen modulates the level of various growth factors. In the present study, the role of transforming growth factor-beta (TGF-beta) in mediating the androgen-regulated growth arrest of LNCaP cells was investigated. The following concentrations of DHT were used: 0, 10(-12), 10(-10), and 10(-7) M. These concentrations were selected because they represent the zero DHT control, the low-proliferative dose, the high-proliferative dose, and the growth-arrest dose, respectively. Results of RT-PCR showed that LNCaP cells express TGF-beta 1 but not -beta 2 and -beta 3 messenger RNA. Competitive quantitative RT-PCR demonstrated that the level of TGF-beta 1 messenger RNA increased approximately 7-fold when cells were treated with 10(-7) M DHT. Results of Western blot analysis showed a dramatic increase in the level of latent TGF-beta 1 protein in cell lysates with increasing concentrations of DHT. In addition, results of enzyme-linked immunoadsorbent assay for TGF-beta 1 indicated that treatment of LNCaP cells with DHT led to a dose-dependent increase in both total and biologically active TGF-beta 1 in the conditioned media. To determine the role of TGF-beta 1 in regulating LNCaP proliferation, the action of TGF-beta 1 was blocked by two different but complementary approaches. First, TGF-beta 1 neutralizing antibody was added to the culture medium with varying concentrations of DHT. Second, mannose-6-phosphate, which has been demonstrated to inhibit the activation of latent TGF-beta 1, was added in a similar manner to the culture. Results demonstrated that the characteristic bell-shaped growth response following treatment with increasing doses of DHT was converted to a linear dose-response curve as the growth inhibition seen at the high dose of DHT was abolished. These observations, taken together, indicate that TGF-beta 1 mediates at least in part the growth arrest observed at the high concentration of DHT in LNCaP cells.