Inhibition of Growth of Human TE2 and C-33A Cells by the Cell-Permeant Calpain Inhibitor Benzyloxycarbonyl-Leu-Leu-Tyr Diazomethyl Ketone

Calpains are Ca2+-requiring, nonlysosomal proteases which are thought to participate in some aspects of intracellular Ca2+-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 μM 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 Ca2+-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 μM ZLVG-CHN2, a cell-permeant inhibitor with little activity against calpains, had decreased Ca2+-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 μM ZLLY-CHN2 in the culture medium. These results indicate that calpains participate in the social regulation of cell growth in multicellular organisms.