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.
Mellgren
R L
RL
Department of Pharmacology and Therapeutics, Medical College of Ohio, Toledo 43699-0008.
Shaw
E
E
Mericle
M T
MT
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