[CANCER RESEARCH 59, 2847–2852, June 15, 1999] Antagonism of p53-dependent Apoptosis by Mitogen Signals 1 Mira Hong, Ming-Derg Lai, Young-Sun Lin, and Ming-Zong Lai 2 Institutes of Molecular Biology [M. H., M-Z. L.] and Biomedical Science [Y-S. L.], Academia Sinica, Taipei 11529; Department of Biochemistry, National Cheng-Kung University, Tainan 70101 [M-D. L.]; Graduate Institute of Microbiology and Immunology, National Yang-Ming University, Taipei 11221 [M-Z. L.]; and Graduate Institute of Immunology, National Taiwan University, Taipei 10002 [M-Z. L.], Taiwan, Republic of China ABSTRACT p53-mediated apoptosis is antagonized by growth factor stimulation. Here, we show that p53-dependent cell death induced by DNA damage was effectively prevented by mitogen activation. The levels of Bcl-2, Bcl-x L , and Bax were not altered by cisplatin treatment and mitogen rescue. Instead, the protection against p53-regulated apoptosis was medi- ated by at least three distinct signaling pathways. Either phosphatidyli- nositol (PI) 3-kinase or mitogen-activated protein kinase kinase (MEK) antagonized p53-induced apoptosis, and an additive preventive effect was observed when both kinases were activated. However, the combination of PI 3-kinase and MEK was not sufficient to completely prevent apoptosis induced by DNA damage. Mitogen activation further suppressed cisplatin- induced p53 expression, and the inhibition was mainly dependent on the Ca 2 pathway. Our results demonstrate that effective antagonism of p53-dependent apoptosis by mitogenic activation requires the presence of multiple signal pathways, including PI 3-kinase, MEK, and Ca 2 . INTRODUCTION DNA damage induces accumulation of p53 followed by apoptosis in many different cells. The accumulation of p53 after DNA damage is mainly due to increased protein stability and enhanced translation (1). The increase of p53 is an essential step in DNA damage-induced apoptosis, as illustrated by the observation that ionizing irradiation does not induce apoptosis in p53 -/- thymocytes (2, 3). p53 stimulates the expression of a number of gene products that are known to participate in the apoptosis process. p53 is a direct activator of the Bax in a few types of cells (4). p53 also stimulates the expression of IGF 3 -binding protein-3 (5), which enhances apoptosis by reducing mitotic signaling (1). A positive correlation between p53-induced expression of GADD45 and apoptosis has also been reported (6). In addition, p53 activates a number of oxidative stress- responding gene products that are involved in the apoptotic process (7). p53-induced apoptosis is blocked by signals initiated from growth factors. p53-mediated apoptosis is suppressed by stimulation with IL-2, IL-3, IL-6, erythropoietin, granulocyte macrophage colony- stimulating factor, and IFN- (6, 8 –14). Cytokines do not affect p53-mediated cell cycle arrest (15); instead, these cytokines prevent p53-regulated apoptosis through distinct mechanisms. For instance, the effect of IL-2 is attributed to the increased expression of Bcl-x L and/or Bcl-2 (13, 14), whereas the action of IL-3 is correlated with the altered expression of p21WAF1/CIP1 and GADD45 (6). PI 3-kinase is well known for its major role in the antiapoptotic signal delivered by growth factors. The activation of PI 3-kinase confers protection from serum withdrawal-induced apoptosis by IGF-1 and nerve growth factor (16 –18). PI 3-kinase also suppresses c-Myc-induced apoptosis (19). The antideath activity of PI 3-kinase is mediated through the activation of Akt/protein kinase B (16, 19, 20). A possible target of Akt is BAD, the phosphorylation of which led to cell survival (21). Despite of the dominant antiapoptotic activity of PI 3-kinase, the direct antagonism of p53-induced death by PI 3-kinase has yet to be demonstrated. In this study, we explored the mechanism of how mitogenic signals antagonize p53-dependent apoptosis induced by cisplatin in trans- formed T cells. For proper mimicking of different mitogenic stimu- lations, combination of protein kinase C activator TPA and calcium ionophore A23187 were used. We demonstrated that the PI 3-kinase pathway directly antagonized p53-induced apoptosis. The effect of PI 3-kinase on the inhibition of p53-dependent apoptosis was further enhanced by coexpression of active MEK1. In addition, mitogen suppressed the p53 accumulation induced by DNA damage, independ- ent of the PI 3-kinase and MEK pathways. Our results suggest that a combination of distinct mitogenic signals is required for effective antagonism of p53-mediated apoptosis. MATERIALS AND METHODS Reagents. Cisplatin, A23187, TPA, and staurosporin were obtained from Sigma Chemical Co. (St. Louis, MO). PD 98059, wortmannin, p38 MAP kinase inhibitor SB203580, and MG 101 (calpain inhibitor I) were purchased from Calbiochem (San Diego, CA). Anti-p53 antibodies (clones PAb421 and PAb1620) were obtained from Oncogene Science (Cambridge, MA). Anti-- tubulin antibody was purchased from Amersham (Buckinghamshire, United Kingdom). Anti-Bcl-2 (N-19) and anti-Bax (P-19) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Bcl-x L antibody was from Transduction Laboratories (Lexington, KY). T-Cell Lines and Treatments. 10I is a T-cell hybridoma that is specific for  repressor cI 12-26 (22). DO11.10 is an ovalbumin 323-329-specific T-cell hybridoma. EL4 T lymphoma (ATCC TIB39; American Type Culture Collection, Manassas, VA) was a gift from Dr. Nan-Shih Liao (Academia Sinica, Taipei, Taiwan, Republic of China). H1299 cell line is p53-null large cell lung carcinoma. Cisplatin was dissolved in water. TPA and A23187 were dissolved in DMSO to make the stock concentration 500-1000 times the final concentration used. The final DMSO concentration in the cultured cells was 0.2% (v/v). The same amount of DMSO was added to the untreated control. Plasmids and Transient Transfection. pCEP4-p53 containing the full- length wild-type p53 in the HindIII site of pSP72 was described previously (23). The constitutively active form of MEK1, pMCL-MEK1-N3/S218E/ S222D (24), was a gift from Dr. Natalie G. Ahn (University of Colorado, Boulder, CO). Wild-type PI 3-kinase p110a was a gift from Dr. Michael D. Waterfield (Ludwig Institute for Cancer Research, London, United Kingdom). Active form of p110 was constructed by introducing a mutation of lysine to glutamic acid at residue 227 according to Rodriguez-Viciana et al. (25). DNAs were transfected into H1299 cells using the calcium phosphate method. Cell Death Measurement. The extent of apoptosis was determined by propidium iodide staining or by annexin V staining, as described previously (26, 27). For cell death involving transient transfection, cells were cotrans- fected with pCMV -gal (Stratagene, La Jolla, CA) or with pGreen Lantern-1 (Life Technologies, Inc., Grand Island, NY). Cells were either stained with 5-bromo-4-chloro-3-indolyl--D-galactopyranoside 72 h after transfection or directly observed under the fluorescence microscope. The numbers of blue, green, and unstained cells were determined by counting six different randomly Received 8/27/98; accepted 4/16/99. 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 1734 solely to indicate this fact. 1 This project was supported by Department of Health Grant DOH87-HR-508, Na- tional Science Council Grant NSC 87-2314-B001-037, and a grant from Academia Sinica (all in Taiwan, Republic of China). 2 To whom requests for reprints should be addressed, at Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan, Republic of China. Phone: (886) 2 2789 9236; Fax: (886) 2 2782 6085. 3 The abbreviations used are: IGF, insulin-like growth factor; IL, interleukin; PI, phosphatidylinositol; MAP, mitogen-activated protein; MEK, MAP kinase kinase; TPA, 12-O-tetradecanoylphorbol-13-acetate. 2847 on May 22, 2016. © 1999 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from