Figure 1. uPA and uPAR protein expression levels and uPA activity correlate with invasive potential of human prostate cancer cell lines.

A. Endogenous uPA and uPAR protein expression was examined by immunoblot analysis of total cellular protein isolated from the following prostate cancer cell lines: LNCaP, DU145 and PC3. Equal amounts of isolated protein from cell extracts of all three cell lines were subjected to immunoblot with anti-uPA, anti-uPAR and anti-GAPDH antibodies. GAPDH was utilized as a loading control.

B. uPA activity in prostate cancer cell lines was assessed by fibrin zymography. Equal amounts of protein from prostate cancer cells in serum-free media were separated by SDS-PAGE on 10% gels containing fibrinogen and plasminogen under non-reducing conditions. After exchange of SDS with Triton X-100 washing, the gel was incubated in glycine buffer (0.1 M, pH 8.0). Fibrinolytic activity was detected as clear lysis bands after amido black staining and subsequent destaining with methanol-acetic acid.

C. Comparison of the in vitro invasive potentials of prostate cancer cell lines. Invasion assays were performed in 12-well transwell chambers containing polycarbonate filters with 12 μm pores coated with matrigel. Cells that had passed to the undersurface of the filters were stained and photographs were taken under microscope at a 200X magnification.

D. Cells invading through the matrigel were counted under a microscope in three random fields at a 200X magnification. Each bar represents the mean ± SD of three fields where significant differences from low or non-metastatic LNCaP cells, which exhibited undetectable uPA and uPAR protein expression, are represented by asterisks * (P <0.05).

(All results are representative of three separate experiments.)