Induction of apoptosis by
tumor necrosis factor (TNF) is modulated by changes 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 microM), a specific inhibitor of
cyclin-dependent kinases (CDK) with high selectivity 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 butyrolactone inhibited the
histone H1 kinase (WEHI, L929 and HeLa) and pRb
kinase (WEHI) activities of CDKs, as compared with the controls. The role of
proteases in the TNF and butyrolactone-induced apoptosis was evaluated by comparing the number and expression of
polypeptides in the cell lysates by gel electrophoresis. TNF and butyrolactone treatment caused the disappearance of several cellular
protein bands in the region between 40-200 kDa, and the 110-90- and 50-kDa
proteins were identified as the major substrates, whose degradation was remarkably increased by the treatments. Interestingly, the loss of several cellular
protein bands was associated with the marked accumulation of two
proteins apparently of 60 and 70 kDa, which may be cleavage products of one or more
proteins. These findings link the decrease of
cyclin-dependent kinase activities to the increase of
protease activities within the growth arrest and apoptosis pathways induced by TNF.