Inhibition of growth of human TE2 and C-33A cells by the cell-permeant calpain inhibitor benzyloxycarbonyl-Leu-Leu-Tyr diazomethyl ketone

Calpains are CA(2+)-requiring, nonlysosomal proteases which are thought to participate in some aspects of intracellular CA(2+)-signal transduction. However, their exact physiologic function has not yet been established. Addition of the cell-permeant, irreversible calpain inhibitor, ZLLY-CHN2, to human TE2 or C-33A cells inhibited growth, as assessed either by mitochondrial MTT reductase activity or by direct cell counting. Inhibition of growth produced by a 24-h exposure to 50 microM ZLLY-CHN2 was reversed upon substituting growth medium without inhibitor. Homogenates produced from cells cultured in the presence of ZLLY-CHN2 displayed decreased calpain and CA(2+)-independent proteolytic activities. Protein immunoblot analysis showed that cell cultures which had lost 80% of their calpain activity still retained full calpain immunoreactivity. Therefore, inhibition by ZLLY-CHN2 appeared to result in accumulation of irreversibly inactivated calpain within the cells. Homogenates from cells cultured in the presence of 20 or 50 microM ZLVG-CHN2, a cell-permeant inhibitor with little activity against calpains, had decreased CA(2+)-independent proteolytic activity, but demonstrated no decrease in calpain activity. ZLVG-CHN2 did not inhibit cell growth under these conditions. Growth of Saccharomyces cerevisiae cells, which do not appear to express calpain-like proteases, was not inhibited by including 50 microM ZLLY-CHN2 in the culture medium. These results indicate that calpains participate in the social regulation of cell growth in multicellular organisms.