Figure 8.

Ca Rac1 Confers Androgen-Independent Growth to LNCaP Cells

A, LNCaP cells stably expressing either the neomycin vector (LNCaP/Neo) or Ca Rac1 (LNCaP/Ca Rac1) were seeded onto 60-mm culture dishes at equal densities and maintained in androgen-depleted media with or without bicalutamide. Tritiated thymidine (1 μCi/ml) was added to cells 48 h after plating, and cells were incubated for an additional 18 h. Cells were then harvested and tritiated-thymidine uptake was monitored as described in Materials and Methods. Data are plotted as average counts per minute (cpm) ± sem. B, LNCaP/Ca Rac1 cells were transfected with AR siRNA or control siRNA using Lipofectamine 2000 for 24 h. Tritiated thymidine (1 μCi/ml) was added to cells 72 h after transfection, and cells were incubated for an additional 18 h. Data are representative of three independent experiments performed in triplicate. Cell lysates were immunoblotted with AR and actin antibodies. C, LNCaP/Neo or LNCaP/Ca Rac1 growth in soft agar was assessed as described in MATERIALS AND METHODS. Average colony number ± sem is plotted. Significance was determined using a two-tailed Student’s t test (*, P < 0.05; **, P < 0.01). D, LNCaP/Ca Rac1 cells (5 × 10⁶) were injected sc into male nude mice. Mice were left intact (n = 8) or castrated (n = 14) 10–14 d after injections (before tumor formation). Tumor volumes were monitored weekly and calculated using the formula L × W × H × 0.53. Data are plotted as average tumor volume ± sem. Cast, Castrated.