Stromal expression of some matrix metalloproteinases (MMPs) has been associated with increasing tumour burden in prostate cancer. We investigated the expression of mRNA (by RT-PCR) and protein (by zymography and western blotting) of MMPs and endogenous inhibitors (tissue inhibitors of metalloproteinases, TIMPs) in two parent epithelial prostate cancer cell lines and sublines of increasing invasive/metastatic potential. Expression of membrane type MMPs, MT1-MMP and MT3-MMP mRNA was higher in PC3-derived than in LNCaPderived lines, whereas MT2-MMP mRNA expression was higher in the LNCaPderived than in PC3-derived cell lines. Active MT1, MT2 and MT3-MMP protein levels were similar in all lines, but processed MT-MMPs, indicative of latent MMP activation, were increased in more aggressive sublines. Expression of MMP-1, MMP-13 and TIMP-1 was higher in the more aggressive sublines and may be implicated in invasive/metastatic ability. Regulation of MMP-1 and MMP-13 expression may offer important therapeutic options for treating patients with prostate cancer.

A considerable problem in proteomics is to separate and identify functional proteins that participate in specific biological processes. To expedite the analysis of active proteases, we have developed a substrate-specific, sensitive in-gel trypsin activity assay after two-dimensional (2-D) separation in a sodium dodecyl sulphate (SDS)-polyacrylamide gel [22]. Using this method, we detected and characterized Arg-specific protease activity in the secreted protein sample of a prostate cancer cell line, PC-3, in 1-D and 2-D gels. Mass spectrometry (MS) identified the protease as urokinase-type plasminogen activator (uPA). Western blotting using anti-uPA antibody and protease inhibition tests confirmed the identification. Since no antibody was involved in the procedure, the result clearly demonstrates the feasibility of this method for identifying novel proteases in biological samples.