REVERSE-ZYMOGRAPHIC ANALYSIS OF PROTEASE NEXIN-II AMYLOID-BETA PROTEIN-PRECURSOR OF HUMAN CARCINOMA CELL-LINES, WITH SPECIAL REFERENCE TO THE GRADE OF DIFFERENTIATION AND METASTATIC PHENOTYPE

Trypsin inhibitors in serum-free conditioned media (SFCM) of various h uman carcinoma cell lines were analyzed by reverse zymography. Most of the cells secreted high-molecular-weight trypsin inhibitors (HMTI) la rger than 100 kDa. The cell lines of colorectal carcinoma origin had a tendency to secrete HMTI whose molecular weight was a little higher t han that of the other cell lines. Analysis of SFCM of subclones with d ifferent histological differentiation and metastatic/invasive potentia ls derived from a single pancreatic carcinoma cell line SUIT-2 showed that the HMTI activity in SFCM was correlated to the degree of histolo gical differentiation in vivo and tended to be inversely correlated to their metastatic/invasive capabilities. Immunoblotting analysis revea led that these HMTI were protease nexin-II/amyloid beta protein precur sors (PN-II/APP). Semi-quantificative reverse-transcriptase/polymerase -chain reaction study for PN-II/APP mRNAs suggested that the differenc es in PN-II/APP activities in SFCM between the subclones might be post -transcriptional or post-secretional events. In addition, SFCM of a hi ghly metastatic subclone contained 43-kDa protein which reacted to ant i-APP monoclonal antibody (MAb) suggesting that the subclone may have APP-degrading activity. (C) 1995 Wiley-Liss, Inc.