TRANSFORMING GROWTH-FACTOR-BETA-1 IS A MEDIATOR OF ANDROGEN-REGULATEDGROWTH ARREST IN AN ANDROGEN-RESPONSIVE PROSTATIC-CANCER CELL-LINE, LNCAP

LNCaP is an androgen-responsive prostatic cancer cell line that exhibi ts a bell-shaped growth response to increasing doses of dihydrotestost erone (DHT) in culture. Although the precise mechanism responsible for this growth response to androgen stimulation remains unclear, many st udies have suggested that androgen modulates the level of various grow th factors. In the present study, the role of transforming growth fact or-beta (TGF-beta) in mediating the androgen-regulated growth arrest o f LNCaP cells was investigated. The following concentrations of DHT we re used: 0, 10(-12), 10(-10), and 10(-7) M. These concentrations were selected because they represent the zero DHT control, the low-prolifer ative dose, the high-proliferative dose, and the growth-arrest dose, r espectively. Results of RT-PCR showed that LNCaP cells express TGF-bet a 1 but not -beta 2 and -beta 3 messenger RNA. Competitive quantitativ e RT-PCR demonstrated that the level of TGF-beta 1 messenger RNA incre ased approximately 7-fold when cells were treated with 10(-7) M DHT. R esults of Western blot analysis showed a dramatic increase in the leve l of latent TGF-beta 1 protein in cell lysates with increasing concent rations 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-bet a 1 in regulating LNCaP proliferation, the action of TGF-beta 1 was bl ocked by two different but complementary approaches. First, TGF-beta 1 neutralizing antibody was added to the culture medium with varying co ncentrations of DHT. Second, mannose-6-phosphate, which has been demon strated to inhibit the activation of latent TGF-beta 1, was added in a similar manner to the culture. Results demonstrated that the characte ristic bell-shaped growth response following treatment with increasing doses of DHT was converted to a linear dose-response curve as the gro wth inhibition seen at the high dose of DHT was abolished. These obser vations, taken together, indicate that TGF-beta 1 mediates at least in part the growth arrest observed at the high concentration of DHT in L NCaP cells.