Search

Cathepsin B and in particular cell-surface and secreted cathepsin B has been implicated in the invasive and metastatic phenotype of numerous types of cancer. We describe here a method to easily survey cancer cell lines for cathepsin B activity using the highly selective substrate Z-Arg-Arg-AMC. Intact human U87 glioma cells hydrolyze Z-Arg-Arg-AMC with a Km of 460 microM at pH 7.0 and 37 degrees C. This is nearly the same as the Km of 430 microM obtained with purified cathepsin B assayed under the same conditions. The pericellular (i.e. both cell-surface and released) cathepsin B activity was inhibited by the cysteine protease inhibitors E-64, leupeptin, Mu-Np2-HphVS-2Np, Mu-Leu-HpHVSPh and the cathepsin B selective inhibitor Mu-Tyr(3,5 I2)-HphVSPh with IC50 values similar to those observed for the inhibition of purified human liver cathepsin B. Other human cancer cell lines with measurable pericellular cathepsin B activity included HT-1080 fibrosarcoma, MiaPaCa pancreatic, PC-3 prostate and HCT-116 colon. Cathepsin B activity correlated with protein levels of cathepsin B as determined by immunoblot analysis. Pericellular cathepsin B activity was also detected in the rat cell lines MatLyLu prostate and Mat B III adenocarcinoma and in the murine lines B16a melanoma and Lewis lung carcinoma. The ability to determine pericellular cathepsin B activity will be useful in selecting appropriate cell lines for use in vivo when analyzing the effects of inhibiting cathepsin B activity on tumor growth and metastasis.

Lapine synovial fibroblasts produce prostaglandin E2 (PGE2) and neutral metalloproteinases in response to phorbol 12-myristate 13-acetate (PMA), human recombinant interleukin-1 (hrIL-1) and, in an autocrine fashion, in response to partially purified preparations of their own cytokines known as cell-activating factors (CAF). Here we have examined the possible role of protein kinase C (PKC) in these responses. Whereas the 80-kDa substrate for PKC could not be detected in synovial fibroblasts, these cells contained a 35-kDa protein which fulfilled the criteria for qualifying as a specific substrate of PKC. Translocation assays based upon phosphorylation of the 35-kDa protein and Western blotting techniques allowed the movement of PKC from the cytosolic to the particulate fraction in response to PMA and CAF to be detected but not in response to 4 alpha-PMA or hrIL-1. Inhibitors of PKC suppressed synovial activation by PMA, partially blocked activation by CAF but had no effect on activation by hrIL-1. There thus appear to be PKC-dependent and PKC-independent routes to synovial cell activation. Our data suggest that IL-1 uses the latter, while CAF contains cytokines which utilize both routes.