[CANCERRESEARCH 57, 5217—5220, December1,1997] Advances in Brief Intracellular Localization of p53 Tumor Suppressor Protein in 7-irradiated Cells Is Cell Cycle Regulated and Determined by the Nucleus' Elena A. Komarova, Carolyn R. Zelnick, Dot Chin, Marija Zeremski, Anatoly S. Gleiberman, Sarah S. Bacus, and Andrei V. Gudkov@ Department of Molecular Genetics, University of Illinois, Chicago, Illinois 60607 (E. A. K., M. Z, A. V. G.J: Advanced Cellular Diagnostics, Elmhurst. Illinois 60126 (C. R. 1, D. C., S. S. B.]; and Department and School ofMedicine, University of California at San Diego. La Jolla, California 92093-0648 (A. S. G) Abstract DNA damage leads to the stabilizationofp53 proteinand its translocation to the nucleus, resulting in activation or suppression ofp53-responsive genes. However, a significant proportion of cell nuclei remain negative for p53 and p53-Inducible cydlin-dependent kinase inhibitor p2i@@a@ after a single dose of ‘y-irradiation. Quantitation of DNA content in p53-positive and -negative nuclei 4-6 h after 10 Gy of ‘y-irradiation of human breast carcinoma MCF7 cells,fibrosarcoma HTiOSOcells,and diploid skin flbroblasts showed that p53 and p2i―@° nuclear accumulation occma predominantly in the G1 phase and at the beginning ofthe S phase ofthe cellcycle@The majority ofthe nuclei in late S phase and in G2-M phase remained p53- and p2i@@a@@negadve. This suggests that there is a cell cycle window during which p53 can accumulate in the nucleus and activate expression of p21'@°. To determine whether cell cyde-dependent distributiOn ofp53 is caused by cytoplasmic modifications of pS3 protein or by properties of the nucleus, p53 localization was analyzed in muhinudeated cells obtained by polyethylene glycol-mediated cell fusion. Dramatic differences in p53 accumulation were found among the nuclei in individual multinudeated cells Distribution of p53-positive and -negative nuclei among the phases of the cell cyde was similar to that observed in a regular cell population. These results suggest that the observed differences in p53 accumulation in the nuclei of irradiated cells are determined by cell cyde-dependent nuclear functions In contrast to p53, p2i'@° was equally distributed among the nuclei ofmuftinudeated cells regardless ofthe stage of the cell cyde, indicating that the observed phenomenon is specific for p53. Introduction Cellular response to different types of stress, including DNA dam age, involves activation of p53 protein that initiates a cascade of events leading to growth arrest or programmed cell death (1). Loss of p53 function in tumor cells occurs in more than half of human malignancies and is associated with high genomic instability, resist ance to chemotherapy and radiation therapy, and metastasis (2). In the organism, p53 is involved in controlling the elimination of genetically damaged cells during early embryogenesis(3) and determinesthe high radiation and drug sensitivity of early embryos as well as several adult tissues with rapid cell turnover (4). The p53-mediated stress response includes three major steps: (a) stabilization of p53 protein; (b) translocation to the nucleus; and (c) activation and suppression of p53-responsive genes (1). p53 activity can be regulated at all of these steps. This regulation may involve either direct modifications of p53 itself or interactions with other cellular proteins. Thus, protein stability can be modulated by viral or Received 8/8/97; accepted 10/I7/97. The costs of publication of this article were defrayed in part by the payment of page charges. l'his article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the National Cancer Institute Grants CA60730 and CA75179 (to A. V. G. and E. A. K.) and United States Army Breast Cancer Research Fellowship (to M.Z.). 2 To whom requests for reprints should be addressed, at Department of Genetics (M/C 669), University of Illinois, 900 South Ashland Avenue, Chicago, IL 60607-7170. Phone: (312) 413-0349; Fax: (312) 996-0683; E-mail: gudkov@uic.edu. cellular factors that affect p53 activity. For example, mdm-2, encoded by a p53-responsive gene, acts as a feedback regulator of p53, causing its rapid degradation (5, 6). To act as a transcription factor, p53 requires interaction with some cellular proteins, including ref- 1 (7), CBP/P300(8, 9), and p33INGI(10). The expressionlevels of such factors may modulate p53-mediated responses by affecting the effi ciency of p53-dependent transcription. In addition, alternatively spliced variants of p53 and different posttranslational modifications (phosphorylation and glycosylation) can also be involved in the reg ulation of p53 stability and function (1, 11). The control of nuclear translocation of p53 is one of the least-studied steps ofp53 regulation. p53 was shown to be actively transported through the nuclear membrane in both directions. Nuclear localization and nuclear export signals have been identified in p53 (12, 13). Nuclear accumulation of wild-type p53 is often blocked in certain types of tumors (including neuroblastomas; Ref. 14), indicating the existence ofan unknown cellular mechanism responsible for p53 nuclear translocation. Differences in the intracellular localization of wild-type p53 protein during the cell cycle under normal growth conditions suggested that such a control mechanism could be cell cycle regulated (15, 16). We have addressed two aspects of the problem of nuclear translo cation of p53. We first showed that nuclear accumulation of p53 after DNA damage and activation of the p53-responsive Wafi/Cipi gene occurred during a specific window of the cell cycle. We then analyzed p53 and p21wafl localization in polynucleated cells to determine how the cell cycle selectivity of nuclear accumulation is regulated. We concluded that it is controlled by nuclear functions rather than by cytoplasmic modifications of p53. Materials and Methods Cell Lines. Breast carcinoma cells (MCF7) and diploid human skin fibro blasts were obtained from American Type Culture Collection. Human fibro sarcoma HT1O8Ocells that express wild-type p53 were provided by George Stark (Cleveland Clinic Foundation, Cleveland, OH). MCF7 cells were grown in RPM! 1640 supplemented with 10% fetal bovine serum and 10 @.tg/ml insulin and maintained in a 10% CO2 humidified atmosphere. Diploid skin fibroblasts and HT1O8O cells were maintained in DMEM with 10 and 20% fetal bovine serum, respectively. Cell Fusion. Cells were plated in 4-well chamber slides (4 x l0@'cells/ chamber) and fused with 50% PEG3solution (M.W.l,450; Sigma) 24—36 h after plating. For fusion, cells were thoroughly washed with PBS, PEG solution was added for 30—40 s, and the cells were immediatelywashed in six changes of PBS. Cell Irradiation. PEG-treated cells were rny-irradiated (10Gy) 2—4h after fusion using a G. L. Shepherd and Associates irradiator, (model 143—68; ‘37Cs source) at a dose rate of 4 Gy/min. Immunohistochemical Analysis. Cells were fixed 4 or 6 h after irradiation and immunostained using anti-p53 (pAbl80l; Calbiochem, San Diego, CA) and anti@p2lW5@ (EAl0@Santa Cruz Biotechnology, Santa Cruz, CA) mono clonal antibodies as described previously ( 17). Double staining for DNA/p53 3 The abbreviation used is: PEG, polyethylene glycol. 5217 on March 21, 2016. © 1997 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from