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 X 10(-2) micrograms 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.