Induction of apoptosis in cancer cells by tumor necrosis factor and butyrolactone, an inhibitor of cyclin-dependent kinases

Induction of apoptosis by tumor necrosis factor (TNF) is modulated by chang es in the expression and activity of several cell cycle regulatory proteins . We examined the effects of TNF (1-100 ng/ml) and butyrolactone I (100 mu M), a specific inhibitor of cyclin-dependent kinases (CDK) with high select ivity for CDK-1 and CDK-2, on three different cancer cell lines: WEHI, L929 and HeLa S3. Both compounds blocked cell growth, but only TNF induced the common events of apoptosis, i.e., chromatin condensation and ladder pattern of DNA fragmentation in these cell lines. The TNF-induced apoptosis events were increased in the presence of butyrolactone. In vitro phosphorylation assays for exogenous histone H1 and endogenous retinoblastoma protein (pRb) in the total cell lysates showed that treatment with both TNF and butyrola ctone inhibited the histone H1 kinase (WEHI, L929 and HeLa) and pRb kinase (WEHI) activities of CDKs, as compared with the controls. The role of prote ases in the TNF and butyrolactone-induced apoptosis was evaluated by compar ing the number and expression of polypeptides in the cell lysates by gel el ectrophoresis. TNF and butyrolactone treatment caused the disappearance of several cellular protein bands in the region between 40-200 kDa, and the 11 0-90- and 50-kDa proteins were identified as the major substrates, whose de gradation was remarkably increased by the treatments. Interestingly, the lo ss of several cellular protein bands was associated with the marked accumul ation of two proteins apparently of 60 and 70 kDa, which may be cleavage pr oducts of one or more proteins. These findings link the decrease of cyclin- dependent kinase activities to the increase of protease activities within t he growth arrest and apoptosis pathways induced by TNF.