Figure 5. Wnt signaling induced by androgen ablation of stromal cells affects epithelial proliferation.

(A) Tgfbr2^{FloxE2/FloxE2} and Tgfbr2^fspKO stromal cells were treated with bicalutamide over five days to analyze Wnt gene expression by semi-quantitative RT-PCR. The expression of 18S ribosomal RNA was used as a loading control. (B) ³H-thymidine incorporation assays were performed on Tgfbr2^{FloxE2/FloxE2} and Tgfbr2^fspKO stromal cells transduced with GFP-adenovirus and grown either in media containing testosterone or bicalutamide (Bic) in the absence of testosterone. ³H-thymidine incorporation assays were performed on LNCaP epithelial cells also treated with or without bicalutamide for five days. Each bar represents the average of three or more replicates and error bars indicate standard deviation. Data is representative of three or more individual experiments. (C) Androgen responsive proliferation of LNCaP cells in response to a 120 hour treatment with conditioned stromal cell media from Tgfbr2^{FloxE2/FloxE2} or Tgfbr2^fspKO for 120 hours as measured by ³H-thymidine incorporation assays. Tgfbr2^{FloxE2/FloxE2} and Tgfbr2^fspKO stromal cells were transduced either with GFP adenovirus as a control or Wnt antagonist SFRP-2 adenovirus prior to starting collection of conditioned media. ‘Bic on Stro’ indicates that bicalutamide was added to stromal cell cultures during the generation of conditioned media. ‘Bic on Epi’ indicates that bicalutamide was added to epithelial LNCaP cells simultaneously with stromal conditioned media treatment. Each bar represents the average of three or more replicates and error bars indicate standard deviation. Compared to the GFP control, p-values for SFRP-2-Tgfbr2^{FloxE2/FloxE2} conditions are 0.207, 0.339, and 0.011, and for SFRP-2-Tgfbr2^fspKO conditions 0.002, 0.005, and 0.004, respectively. Each asterisk indicates statistical significance between the control and knockout data points for a given condition with a p-value less than 0.05.