ROLE OF CASPASES (ICE CED-3 PROTEASES) IN DNA-DAMAGE AND CELL-DEATH IN RESPONSE TO A MITOCHONDRIAL INHIBITOR, ANTIMYCIN-A/

Caspases (ICE/Ced3 proteases) are closely related family of cysteine p roteases that play a key role in apoptotic cell death. We examined the role of caspases in DNA damage and cell death in response to the mito chondrial inhibitor, antimycin A. LLC-PK1 cells contain caspase activi ty that was markedly inhibited by cleavage site-based peptide inhibito rs of caspases but not by inhibitors of serine, cysteine, aspartate or metalloproteinases. The caspase activity increased within five minute s of exposure to antimycin A, preceding any evidence of DNA damage and cell death. The specific caspase inhibitors, Ac-Tyr-Val-Ala-Asp-aldeh yde (inhibitor I) and Ac-Asp-Glu-Val-Asp-aldehyde (inhibitor II) preve nted, in a dose dependent manner, antimycin A-induced DNA strand break s as determined by DNA unwinding assay (residual double stranded DNA i n control, 94 +/- 2%; antimycin A alone, 48 +/- 3%; antimycin A + inhi bitor I at 50 mu M; 93 +/- 2%; antimycin A + inhibitor II at 50 mu M 8 9 +/- 5%; N = 3 to 4, P < 0.001). These inhibitors also prevented anti mycin A-induced DNA fragmentation as determined by agarose gel electro phoresis and by in situ labeling of cell nuclei by the terminal deoxyn ucleotidyl transferase (TdT) nick end labeling (TUNEL) method. The cas pase inhibitors markedly prevented antimycin A-induced cell death in a dose-dependent manner as measured by trypan blue exclusion (control 6 +/- 1%, antimycin A alone 40 +/- 1%, antimycin A + inhibitor I at 50 mu M 16 +/- 1%; N = 4 to 7, P < 0.001). These data indicate that the c aspase family of enzymes play an important role in DNA damage and cell death in response to the mitochondrial inhibitor, antimycin A.