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

Brazilian Journal of Medical and Biological Research (1999) 32: 473-482 Induction of apoptosis by TNF and butyrolactone ISSN 0100-879X 473 Induction of apoptosis in cancer cells by tumor necrosis factor and butyrolactone, an inhibitor of cyclin-dependent kinases J.E. Belizário1,2, S. Sherwood3 and W. Beçak1 1Laboratório de Genética, Instituto Butantan, São Paulo, SP, Brasil 2Departamento de Farmacologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brasil 3Genentech, Inc., South San Francisco, CA, USA Abstract Correspondence J.E. Belizário Departamento de Farmacologia ICB, USP 05508-900 São Paulo, SP Brasil E-mail: Research supported by FAPESP (Nos. 93/0327-4 and 96/0860-6) and CNPq (Nos. 400187-93 and 300786/94-8). Received May 20, 1998 Accepted December 21, 1998 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 µM), 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 butyrolactoneinduced 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 11090- 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. Key words · Apoptosis · Cell cycle · Cyclin-dependent kinases · Cyclin-dependent kinase chemical inhibitors · Tumor necrosis factor Braz J Med Biol Res 32(4) 1999 474 J.E. Belizário et al. Introduction Tumor necrosis factor-a (TNF-a) is a 17-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). Apoptosis is a selective program for cell death controlled by specific genes, which either suppress (bcl-2/CED-9 protein family) or promote it (CED-3/ICE protein family) (3,4). The molecular and morphological events triggered by these regulatory proteins lead to the internucleosomal fragmentation of DNA, degeneration of nuclear and cytoplasmic structures and formation of membrane-bound apoptotic bodies, which are engulfed by neighboring cells or tissue macrophages (5,6). Recently, a network of genes, including various cell cycle genes, protooncogenes, tumor suppressor genes and cell death genes, was shown to play major roles on the regulation of cell growth, differentiation and apoptosis as well as in tumor progression (7-9). Two interrelated cellular processes, 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 (11-13) and a concomitant increase of the tumor suppressor protein p53 and the CDK inhibitory protein, p21 (14-16). Furthermore, the expression of both cyclin D3 and c-myc can sensitize cancer cells to TNF-induced apoptosis (17). Molecular interaction-based screens have revealed molecules derived from microorganisms, plants and animals which inhibit the activity of the cyclin-dependent kinases (CDK-1 to 7) and their regulatory catalytic Braz J Med Biol Res 32(4) 1999 subunits, cyclins A to H (18,19). Butyrolactone I is a microbial alkaloid isolated from an Aspergillus strain (20) which is a competitive inhibitor of ATP binding to the ATPbinding pocket of the CDKs. Butyrolactone is a highly selective inhibitor of CDK-1 and CDK-2 in vitro and was shown to arrest the normal and cancer cell cycle progression from the G1 to the S phase and from the G2 to the M phase (21). Moreover, it suppresses the phosphorylation of pRb at the G1-S phase and promotes apoptosis of HL-60 cells at doses of 20-50 µM (22). The present data show that the induction by TNF of both chromatin condensation and DNA fragmentation in the cancer cell lines WEHI, L929 and HeLa S3 is increased in the presence of butyrolactone. These effects were accompanied by the inhibiton of histone H1 and pRb kinase activities as well as by the proteolysis of several cellular proteins in the apoptotic cells compared with untreated control cells. Material and Methods Cell culture and drug treatment The cell lines WEHI, methylcholanthreneinduced mouse fibrosarcoma (CRL 1751), L929, mouse fibrosarcoma (CCL 1), and HeLa S3, human cervix carcinoma (CCL 2.2) were obtained from the Americam Type Culture Collection. The cells were maintained in DMEM, 10% fetal bovine serum, 2 mM glutamine and antibiotics in a 5% CO2 incubator. The cells were separated using trypsin/EDTA solution and harvested by centrifugation at 800 g for 5 min. For the cytotoxic assays, cells were incubated with complete medium with or without TNF (1-100 ng/ml) of human or mouse origin. Recombinant mouse and human TNF were provided by Genentech, Inc. (South San Francisco, CA, USA). At the time indicated, the dead cells and adherent cell populations were harvested by centrifugation, washed with PBS and divided into aliquots for the morpho- 475 Induction of apoptosis by TNF and butyrolactone logical and biochemical assays. The effects of butyrolactone I, a selective inhibitor of CDK-1 and CDK-2 kinase activity (20,21), on cell growth and apoptosis were determined by incubating the cell lines with 100 µM of the drug for 1 h following the addition of TNF and by further incubation for 1-24 h. Butyrolactone was provided by Dr. Akira Okuyama (Banyu Tsukuba Research Institute and Merck Research Labs, (...truncated)