Abstract
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.
Highlights
Tumor necrosis factor-a (TNF-a) is a17-kDa protein primarily produced by macrophages with a wide range of biological activities [1]
Evidence in a number of studies has suggested that oncoproteins, tumor suppressor proteins and the cell cycle regulatory proteins play a critical role in the mechanism by which normal and tumor cells respond to
Several chemical inhibitors of cyclin-dependent kinases (CDK) have been found on the basis of their ability to inhibit cell cycle proliferation and/or to induce apoptosis in cancer cells [18,19]
Summary
Tumor necrosis factor-a (TNF-a) is a17-kDa protein primarily produced by macrophages with a wide range of biological activities [1]. TNF exerts cytotoxic or cytostatic effects on a variety of cell types, which may result in cell death by apoptosis [2]. The cell cycle and cell death, are involved simultaneously during the cellular response to TNF, with some cell types showing that growth arrest was accompanied by apoptosis. The growth inhibitory effects of TNF in normal and cancer cells have been associated with a G1 phase arrest [10] and a decrease in the activity of cyclin-dependent kinases (CDKs) [11], the inhibition of the expression of cyclin A, cyclin B [11,12,13] and a concomitant increase of the tumor suppressor protein p53 and the CDK inhibitory protein, p21 [14,15,16]. The expression of both cyclin D3 and c-myc can sensitize cancer cells to TNF-induced apoptosis [17]
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