Wild-Type p 53 Can Induce p 21 and Apoptosis in Neuroblastoma Cells But the DNA Damage-induced G 1 Checkpoint Function Is Attenuated 1

p53 is a tumor suppressor protein important in the regulation of apoptosis. Because p53 functions as a transcription factor, cellular responses depend upon activity of p53 localized in the nucleus. Cytoplasmic sequestration of p53 has been proposed as a mechanism by which the function of this protein can be suppressed, particularly in tumor types such as neuroblastoma in which the frequency of mutations of p53 is low. Data presented here demonstrate that nuclear p53 protein is expressed in a panel of neuroblastoma cell lines, and after exposure to DNA damage, transcriptionally active p53 expression can be induced. After exposure to both equitoxic IC80 and 10-Gy doses of ionizing radiation, both p53 and p21 were induced, but G1 cell cycle arrest was attenuated. To investigate whether the DNA damage signaling pathway was incapable of inducing sufficient p53 in these cells, we expressed additional wild-type p53 after adenoviral vector transduction. This exogenous p53 expression also resulted in p21 induction but was unable to enhance the G1 arrest, suggesting that the pathway downstream from p53 is nonfunctional. Although p53-mediated G1 arrest is attenuated in neuroblastoma cells, the ability of p53 to induce apoptosis appears functional, consistent with its chemosensitive phenotype. This work demonstrates that p53 is expressed in the nucleus of neuroblastoma cells and can mediate induction of p21. However, this cell type appears to have an attenuated ability to mediate a DNA damage-induced G1 cell cycle arrest. INTRODUCTION p53 protein has been described as the “guardian of the genome” because it has the ability to induce apoptosis when either DNA is damaged or cell growth is deregulated (1). In this way, p53 acts as a tumor suppressor by activating an apoptotic pathway in cells that may be accumulating mutations or proliferating abnormally. The mechanism by which p53-dependent apoptosis occurs is not completely understood, but it appears to be mediated by the ability of p53 to bind DNA and act both as a positive and negative regulator of transcription (2). After DNA damage, p53 activates transcription of the cdk 4 inhibitor p21, which mediates a growth-inhibitory function (3). Cells then arrest in the G1 phase of the cell cycle until either DNA damage has been repaired or apoptosis is induced (1, 3). As a consequence, cells that express a functional wtp53 and have the ability to induce p53-dependent apoptosis are generally more sensitive to the action of DNA-damaging agents (4–6). The p53 gene is the most frequently mutated gene in human cancer (7), and p53-deficient mice have a high frequency of spontaneous tumor formation (8), consistent with the function of p53 as a tumor suppressor. The mutation frequency of p53 varies among tumor histiotypes, ranging from 0 to 5% in NBs (9) to 75–80% in colon carcinomas (7); however, even if the gene itself is not mutated, the function of wtp53 protein or another protein in a p53-activated cascade may be attenuated. One of the best characterized mechanisms by which wtp53 function can be inhibited is by the binding of p53 protein to MDM2 (10, 11). More recently, cytoplasmic sequestration of wtp53 protein has also been suggested as a mechanism by which its function can be attenuated (12, 13). NBs are pediatric tumors derived from neural crest cells of the sympathetic nervous system (14). They generally respond well to initial chemotherapeutic regimens and are therefore classed as a chemosensitive tumor type (15). Consequently, patients that present with local or regional disease have an excellent prognosis with a survival rate of 80–100% (14). As described above, NBs usually contain a wtp53 gene (9), an observation consistent with their chemosensitive phenotype (15). However, it is presently controversial as to whether p53 is functional in this tumor type. Moll et al. (13) have reported that p53 protein is sequestered in the cytoplasm of NBs and that the DNA damage-induced G 1 arrest phenotype is attenuated. In contrast, Goldmanet al. (16) have reported both nuclear and Received 5/25/99; revised 8/23/99; accepted 8/31/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by NIH Grants CA77541, CA23099, and the Cancer Center Support Grant CA21765; by the American Lebanese Syrian Associated Charities; and the Association pour la Recherche sur le Cancer. 2 Present address: Laboratoire de Pharmacologie, Institut Claudius Regaud, 20–24, rue du Pont St. Pierre, Toulouse Cedex, France 31052. 3 To whom requests for reprints should be addressed, at Department of Molecular Pharmacology, St. Jude Children’s Hospital, 332 North Lauderdale, Memphis, TN 38015. Phone: (901) 495-3833; Fax: (901) 5211668; E-mail: linda.harris@stjude.org. 4 The abbreviations used are: cdk, cyclin-dependent kinase; wtp53, wild-type p53; mp53, mutant p53; IR, ionizing radiation; TDN, transdominant negative; SJCRH, St. Jude Children’s Research Hospital; HRP, horseradish peroxidase; TUNEL, TdT-mediated dUTP nick end labeling; NB, neuroblastoma. 4199 Vol. 5, 4199–4207, December 1999 Clinical Cancer Research Research. on October 16, 2017. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from cytoplasmic p53 localization and an intact G 1 arrest after DNA damage. Using highly sensitive immunofluorescence immunohistochemistry and equitoxic doses of IR, we have demonstrated that p53 is present in the nucleus of NB cells and that it is transcriptionally active. In addition, although the ability of these cells to induce p53-mediated apoptosis is functional, the p53mediated G1 arrest pathway is attenuated. MATERIALS AND METHODS Cell Lines. The NB cell lines SJNB-1 and SJNB-4 were derived at SJCRH; cell lines NB-1643 and NB-1691 were obtained from the Pediatric Oncology Group cell bank (17). The Rh30 rhabdomyosarcoma cell line, obtained from Dr. Peter Houghton, was also derived from a SJCRH patient tumor. ML-1 myeloid leukemia cells were obtained from Dr. Gerard Zambetti (SJCRH). These cell lines were cultured in RPMI 1640 (BioWhitaker, Walkersville, MD) supplemented with 10% FCS (Hyclone, Logan, UT) and incubated at 37°C in a humidified atmosphere of 5% CO 2, 95% air. Nonessential amino acids (Life Technologies, Inc., Grand Island, NY) were added to medium used to grow ML-1 cells. The stably transfected NB-1643 cells, designated NB-1643p53 -1 (p53-1) were maintained in the presence of 250 mg/ml Geneticin (Life Technologies, Inc.). SK-N-SH cells and IMR32 cells, obtained from the American Type Culture Collection (Rockville, MD), were cultured in DMEM medium (BioWhitaker) also containing 10% FCS (Hyclone) and incubated at 10% CO 2/90% air. Antibodies. The p53 monoclonal antibodies used in the indirect immunofluorescence studies were DO7 and BP53.12. These antibodies were obtained from PharMingen (San Diego, CA) and Oncogene Research Products (Cambridge, MA), respectively. The isotype-matched control antibodies were IgG 2b and IgG2a, respectively (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Primary antibodies used in the Western blot analyses were p21 (C-19), p53 DO1-HRP-conjugated antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and a monoclonal antib tubulin antibody from Sigma Chemical Co. (St. Louis, MO). Adenoviral Vector and Transduction. The p53 adenoviral vector Ad.p53 was obtained from Genetic Therapy, Inc., a Novartis Company (Gaithersburg, MD). Cells were transduced (multiplicity of infection 10) for 2 h inmedium containing 2% FCS (Hyclone), after which time complete medium (10% serum) was added. The virus was removed, and fresh medium was added at 24 h. Indirect Immunofluorescence Stain. These studies were performed as described previously (18) with minor modifications. Briefly, cells were grown on glass chamber well slides (Nunc, Naperville, IL) and fixed with either freshly prepared 1% paraformaldehyde at room temperature or acetone: methanol (1:1) at 220°C for 2 min. All subsequent procedures were carried out at room temperature. When cells had been fixed, nuclear membranes were permeabilized with 0.25% Triton in PBS for 20 min. Cells were washed three times in PBS and then incubated with 10% swine serum for 20 min to prevent nonspecific binding of antibodies. Incubation with the primary antibody or an isotype-matched control was fo r 2 h in ahumidified chamber. Cells were then washed with PBS for 10 min, changing the wash solution five times during this period. The cells were then incubated with a FITC-conjugated donkey antimouse IgG from Jackson ImmunoResearch Laboratories, Inc. for 1 h. After washing in PBS for 10 min, cells were incubated with 30 mM Hoechst 33342 for 3 min to stain the DNA. The slides were air dried and mounted. Fluorescence Image Cytometry. To eliminate investigator bias, fields of cells to be analyzed for p53 content were chosen solely on the basis of Hoechst (DNA) fluorescence. DNA fluorescence was used to confirm that nuclei were intact and in a single focal plane. DNA images of cells meeting these criteria were acquired and stored for analysis as described previously (18). The microscope was then reconfigured to visualize p53 FITC fluorescence. Images of FITC fluorescence were acquired using 5-s exposures and stored for subsequent analysis. Cells were analyzed on a Zeiss Axiovert 135TV microscope connected to a Silicon Graphics Crimson workstation as described previously (18). Photomicrographs were printed using linear contrast. The nonspecific electronic background fluorescence introduced by the camera to the microscopic field was subtracted prior to printing. Preparations of Soluble Cellular Proteins. Extracts of soluble cellular protein were prepared by resuspending cell pellets in extract buffer [50 mM Tris-HCl (pH 8.0), 0.