p34cdc2 Kinase Activation and Apoptosis Inhibited by I 2-O-Tetradecanoylphorbol-1 3-acetate in Human Breast MCF-7 Carcinoma CeHs1

The p34 kinase is a highly regulated serinethreonine kinase that when complexed with cyclins A and B, controls cell entry Into mitosis. Recently, premature activation of p34 was shown to be required for apoptosis induced by a wide variety of agents. Here, we show that Taxol induced p34 kinase activity with a peak at 6 h In human breast carcinoma MCF-7 cells. We subsequently observed that the activation of CPP32/Yama protease as well as the cleavage of its substrate poly(ADP-ribose) polymerase occurred 9 h after Taxol treatment Olomoucine, a potent p34#{176} inhibitor, effectively prevented Taxol-Induced p34 ( kinase activation and subsequent apoptosis. Furthermore, the treatment of cells with cydlin BI-specific antisense oligonucleotide also blocked Taxol-induced apoptosis, suggesting that cyclin BI-associated p34 kinase plays an important role in the Induction of apoptosis by Taxoi 12-0Tetradecanoylphorbol-13-acetate (TPA , a protein kinase C activator, was found to exert strong protection against Taxol-Induced cell death in MCF-7 cells. WA Inhibited Taxol-medlated activation of p34cdc2 kinase by preventing the dephosphorylatlon of the Tyr-15 residue on p34#{176} without aftering the levels of Cdc2 and cyclin BI. In contrast, the ability of Taxol to enhance tubulin polymerization was not inhibited by TPA. These findings suggest that modulation of protein kinase C signaling can protect against Taxol-induced cell death by inhibiting p34 kinase activation. Received 7/23/97; revised 9/30/97; accepted iO/3i/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement In accordance with 18 U.S.C. Section i734 solely to mdicate this fact. 1 Supported by National Science CouncIl Grant NSC 86-2621-B002005-z. 2 To whom requests for reprints should be addressed, at Laboratory of Molecular & Cellular Toxicology, Institute of Toxicology, Collage of Medicine, National Taiwan University, No. 1 , Sec. 1, Jen-Al Road, Taipei, Taiwan. Fax: 011-886-2-341-0217. Introduction Paclitaxel (Taxol) is an effective drug that shows encouraging activity in human ovarian and metastatic breast cancers (1) and malignant melanoma (2). The drug is an antimitotic agent whose action results in the formation of stable bundles of microtubules within cells (3). These effects of the drug are correlated with causation of mitotic and G2-M arrest as well as cellular toxicity (4, 5). Although the interaction of Taxol with the cytoskeleton is well characterized, the molecular mechanisms by which such an interaction leads to cell cycle arrest and cell death are not well understood. The p34 kinase is universally required as the master control enzyme during mitosis in eukaryotes (6). Its activity controls the G2-M-phase transition by promoting breakdown of the nuclear membrane, chromatin condensation, and microtubule spindle formation. This kinase is activated by Thr1 61 phosphorylation and its association with other proteins, primarily cyclin B. During G2, Cdc25 phosphatase binds to and activates by cyclin B/Cdc2 complex by dephosphoryIating Cdc2 at phosphotyrosine 15 and phosphothreonine 14 residues, thus allowing catalysis of ATP within an ATP binding pocket (7, 8). Exit from mitosis requires the inactivation of p34cdc2 by degradation of the cyclin B-regulatory subunit (9) and dephosphorylation of p34 at Thr-161 (10). Recently, premature activation of p34 2 was reported to be required for apoptosis induced by a lymphocyte granule protease (1 1). An unscheduled and transient P34 activation was found in the apoptosis of HL-60 cells treated with anticancer drugs such as etoposide, camptothecin, and nitrogen mustard (12). Furthermore, treatment of HeLa cells with Taxol caused p34cdc2 activation that did not occur at the mitotic block but rather coincided with DNA fragmentation (13). These data strongly indicate that inappropriate activation of p34CciC2 may play an important role in Taxol-mediated apoptosis. TPA,3 an activator of PKC, was capable of modulating apoptosis induced by a wide variety of agents. TPA attenuated the expression of p53 as well as the induction of apoptosis in wild-type p53-transfected K562 cells treated with doxorubicin (14). In contrast, IRA treatment induced apoptotic cell death in androgen-sensitive human prostate LNCaP cells and human leukemic HL-60 cells (1 5, 1 6). These suggest that the critical point determining the fate of cellular response to WA seems to depend on the type of cell and its state of differentiation or growth. However, the mechanisms 3 The abbreviations used are: WA, 12-O-tetradecanoylphorbol-13-acetate; PARP, poly(ADP-ribose) polymerase; PKC, protein Kinase C; ICE, interleukln ip-converting enzyme; Ac-DEVD-AMC, acetyl-Asp-Glu-ValAsp-aminomethyl cournarin.