Inhibition of caspase-3-like activity prevents apoptosis while retaining functionality of human chondrocytes in vitro

Apoptosis was induced in a human chondrocyte cell line, T/C 28a4, by treatm ent with various stimuli, including camptothecin, tumor necrosis factor-alp ha, staurosporine, okadaic acid, and reduced serum conditions. All stimuli induced a cytosolic DEVDase activity, coincident with apoptosis. Caspase ac tivities in the lysates were characterized and quantitated with peptide cle avage profiles. To confirm that the results were not related to the immorta lized nature of the cell line, primary human chondrocytes also were shown t o undergo apoptosis under similar conditions, which resulted in increased c ytosolic DEVDase activity. There was little or no caspase-1 (interleukin-1 beta-converting enzyme) or caspase-8-like activity in the apoptotic cells. In all cases, the irreversible nonselective caspase inhibitor, 2-VAD-FMK, a nd the caspase-3-selective inhibitor, Ac-DMQD-CHO, inhibited DEVDase activi ty and apoptosis, whereas the caspase-1-selective inhibitor, Ac-YVAD-CHO, h ad no effect. Human chondrocytes were stably and transiently transfected wi th a type-II collagen gene (COL2A1) regulatory sequence driving a luciferas e reporter as a specific marker of chondrocyte gene expression. Treatment o f the cells with camptothecin or tumor necrosis factor-alpha plus cyclohexi mide significantly inhibited COL2A1 transcriptional activity. Significantly , cotreatment with Z-VAD-FMK or AcDMQD-CHO maintained COL2A1-reporter gene activity, indicating that the prevention of apoptosis by caspase-3 inhibiti on was sufficient to maintain cell functionality as assessed by the retenti on of type-II collagen promoter activity.