Protease activity associated with loss of adhesiveness in mouse teratocarcinoma

We have observed the spontaneous conversion of an embryoid body (multicellular) form of 129/J mouse ascites teratocarcinoma to a single cell form. Concomitant with the conversion, a rapid increase in growth rate and ascites fluid accumulation have been observed. This report presents data on protease activity in the ascites fluid after the conversion and evidence that the protease causes decreased tumor cell adhesiveness. Ascites protease has a pH optimum of 2.0-3.0 and is inhibited by both DAN (diazoacetyl-dl-norleucine methyl ester) and Pepstatin A. Using denatured bovine hemoglobin as a substrate, the low pH optimum and inhibition by DAN and Pepstatin A allow tentative identification of the enzyme as the carboxypeptidase Cathepsin D. Approx. 0.036 × 10^-2 μg of Cathepsin D per mg of protein was found in the ascites fluid of the single cell form of the mouse teratocarcinoma. We show that Pepstatin A-derivatized agarose beads bind the single ascites cells, causing them to display increased cell-cell adhesion, a phenomenon not observed with control beads. The results suggest that ascites protease may play a role in transformation of a slow growing, clustered tumor into a rapidly growing, non-adhesive, single cell form. We found that an embryoid-like tissue culture line of mouse teratocarcinoma, that we established, disaggregated into single cells, upon addition of ascites fluid from the single cell tumor, to the culture medium. Pepstatin A prevented disaggregation of the cell clusters. These results further support the contention that specific ascites protease plays a role in the transformation of a clustered tumor into a single cell form.