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.
Department of Pharmacology and Therapeutics, Medical College of Ohio, Toledo 43699-0008.
R L
RL
Mellgren
E
E
Shaw
M T
MT
Mericle
D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects*; D002461:Cell Line, Transformed; D002463:Cell Membrane Permeability; D002472:Cell Transformation, Viral; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism; D003978:Diazomethane / Q000031:analogs & derivatives* / Q000494:pharmacology; D005260:Female; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D012441:Saccharomyces cerevisiae / Q000187:drug effects; D013539:Simian virus 40 / Q000235:genetics; D013997:Time Factors; D002583:Uterine Cervical Neoplasms
D002135:Calcium-Binding Proteins; D009097:Multienzyme Complexes; D009842:Oligopeptides; D011480:Protease Inhibitors; C090218:benzyloxycarbonylleucyl-leucyl-tyrosine diazomethyl ketone; D003978:Diazomethane; C033668:calpastatin; D002154:Calpain; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex