No abstract

~,-Antit~psin; (a,-AT) produced by various human carcinoma (non-hepatoma) cell lines were analyzed. Ftve out of eight ceil lines secreted detectable amounts of a,-AT into the ~onditloned media. All were aden~arcinoma cell lines. The tumor cell-derived a,-ATs had higher molecular weights (MW) than the normal plasma form. Most of this difference was an overall reflection of altered ~-~ycosylation. As judged by binding of iectins, the glycosyiation had shifted towards higher levels of triantennary oligosaccharides and higher levels of fucosylation. The condittoned media also contained lower MW

Trypsin inhibitors in serum-free conditioned media (SFCM) of various human carcinoma cell lines were analyzed by reverse zymography. Most of the cells secreted high-molecular-weight trypsin inhibitors (HMTI) larger than 100 kDa. The cell lines of colorectal carcinoma origin had a tendency to secrete HMTI whose molecular weight was a little higher than that of the other cell lines. Analysis of SFCM of subclones with different histological differentiation and metastatic/invasive potentials derived from a single pancreatic carcinoma cell line SUIT-2 showed that the HMTI activity in SFCM was correlated to the degree of histological differentiation in vivo and tended to be inversely correlated to their metastatic/invasive capabilities. Immunoblotting analysis revealed that these HMTI were protease nexin-II/amyloid beta protein precursors (PN-II/APP). Semi-quantificative reverse-transcriptase/polymerase-chain reaction study for PN-II/APP mRNAs suggested that the differences in PN-II/APP activities in SFCM between the subclones might be post-transcriptional or post-secretional events. In addition, SFCM of a highly metastatic subclone contained 43-kDa protein which reacted to anti-APP monoclonal antibody (MAb) suggesting that the subclone may have APP-degrading activity.

A new human cell line, LC‐2/ad was established from pleural effusion of pulmonary adenocarcinoma of a 51 year old Japanese female. The LC‐2/ad cells exhibit an epithelial appearance and a tendency to form small domes as observed with phase‐contrast microscopy. The modal chromosome number was 53–56. Plating efficiency and doubling time were 6.8% and 58 h, respectively (32th passage). Immunocytochemlcally, the cells were strongly positive for CEA and cytokeratins including cytokeratin no. 18 which is present in simple epithella. Ultrastructurally, the cultured cells were characterized by well‐formed junctional complexes and microvilli. Subcutaneous injection of 5 × 106 cells into a nude mouse resulted in tumor formation classified histologically as a moderately differentiated adenocarcinoma. This cell line produced at least two functionally active trypsin inhibitors together with several proteinases in vitro. The main inhibitor was purified partially from the serum‐free conditioned medium and confirmed immunologically as human α1‐antitrypsin (AAT). Immunohistochemically, the xenografted tumor was also positive for AAT. The cell line LC‐2/ad is useful for the study of tumor‐derived serine proteinase inhibitors, in particular AAT.

Trypsin inhibitors secreted by human colorectal adenocarcinoma cell lines were analyzed by reverse zymography. Among eleven cell lines analyzed, the major inhibitor secreted was protease nexin-II (PN-II), a secreted form of amyloid beta protein precursor (APP) containing a Kunitz-type serine proteinase inhibitor domain. Expression of the APP gene was also confirmed in the cell lines and the main APP mRNA species were PN-II types. The APP gene expression was constant during cell growth in vitro. On the other hand, the rate of extracellular PN-II accumulation markedly increased after long-term serum-free maintenance of the confluent culture. The extracellular accumulation of PN-II was also strongly stimulated either by interleukin-1beta (IL-1beta) treatment or to a lesser extent by basic fibroblast growth factor, tumor necrosis factor-alpha, hepatocyte growth factor or epidermal growth factor. Neither serum depletion- nor IL-1beta-induced stimulation of extracellular PN-II accumulation were accompanied by obvious alteration of the levels of APP mRNA and cellular APP holoprotein, suggesting that the enhanced extracellular accumulation of PN-II might result from up-regulation of the secretory pathway of APP. The IL-1beta-induced PN-II secretion was significantly inhibited by relatively high concentrations (50-200 microg/ml) of aprotinin, a serine proteinase inhibitor, in a dose-dependent manner without obvious cell-toxic effects.