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P53 Activation in Chronic Radiation-Treated Breast Cancer Cells: Regulation of MDM2/P14arf

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P53 Activation in Chronic Radiation-Treated Breast Cancer Cells: Regulation of MDM2/P14arf [CANCER RESEARCH 64, 221–228, January 1, 2004] p53 Activation in Chronic Radiation-Treated Breast Cancer Cells: Regulation of MDM2/p14ARF Liqun Xia, Aimee Paik, and Jian Jian Li Radiation Biology, Division of Radiation Oncology, Beckman Research Institute, City of Hope National Medical Center, Duarte, California ABSTRACT than 100 genes of human genome have been identified as have binding sites for p53 (22), and many stress genes activated by IR are Mammalian cells chronically exposed to ionizing radiation (IR) induce a result of p53 activation (23, 24). With a link to these IR effector stress response with a tolerance to the subsequent cytotoxicity of IR. genes, p53 activity has been shown to affect the anticancer efficiency Although p53 is well documented in IR response, the signaling network causing p53 activation in chronic IR remains to be identified. Using breast of radiotherapy using FIR (25). Additional evidence suggest that p53 carcinoma MCF؉FIR cells that showed a transient radioresistance after is very sensitive to IR-induced DNA damages (26), and DNA strand exposure chronically to fractionated IR (FIR), the present study shows breaks induced by IR are believed to be the major source that trigger that the basal DNA binding and transcriptional activity of p53 was the p53-dependent repair system (27). However, it is unclear how p53 elevated by FIR. p53-controlled luciferase activity was strikingly induced is regulated in cells that showed a transient tolerance to IR after (ϳ7.9-fold) with little enhancement of p53/DNA binding activity (ϳ1.3- exposure chronically to IR. This transient radioresistant phenotype in fold). The phosphorylated p53 (Thr 55) was increased in the cytoplasm radiation-derived cells (8–10) presents a tolerance of tumor cells to .and nucleus of MCF؉FIR but not in the sham-FIR control cells. On the radiotherapy contrary, the sham-FIR control MCF-7 cells showed a low p53 luciferase Both MDM2 functioning in p53 protein degradation and p14ARF transcription (ϳ3-fold) but a striking enhancement of p53/DNA binding functioning in MDM2 inhibition may be involved in the metabolic (12-fold) after 5 Gy of IR. To determine the signaling elements regulating p53 activity, DNA microarray of MCF؉FIR using sham-FIR MCF-7 cells regulation of p53 activity (28, 29). IR-induced p53 initiates the as a reference demonstrated that the mRNA of p21, MDM2, and p14ARF signaling process that causes cell cycle arrest, which is accompanied was up-regulated. Time course Western blot analysis, however, showed no by the ability for DNA repair and apoptosis (30, 31). This process can difference in p21 induction. In contrast, MDM2 that was absent in control be actively regulated by expression of MDM2. Activation of p53 and ؉ cells and was predominantly induced by IR was not induced in MCF FIR MDM2 is found in cells arrested at G1-S phase or G2-M boundaries cells. In agreement with MDM2 inhibition, MDM2-inhibitory protein induced by IR (17). Interaction of MDM2 with the p53 NH2-terminal p14ARF was increased in MCF؉FIR cells. In summary, these results region has been shown to accelerate p53 degradation and to inhibit the demonstrate that up-regulation of p14ARF paralleled with MDM2 inhi- capacity for p53-mediated gene transcription (28, 29). In addition, bition contributes to p53 accumulation in the nucleus and causes a high MDM2 is found actively involved in the process of p53 nuclear responsiveness of p53 in chronic IR-treated breast cancer cells. exclusion (32), and the sequence required for MDM2-mediated p53 degradation and nuclear export has been identified (33, 34). MDM2 is INTRODUCTION also shown to be a unique ubiquitin-protein ligase to ubiquitinate and degrade p53 (28, 29). Mammalian cells treated with ionizing radiation (IR) induces a However, the inhibitory effect of MDM2 on p53 can be counter- stress response associated with an enhanced tolerance to the subse- acted by p14ARF, a protein that directly binds to MDM2 and, as a quent cytotoxicity of radiation (1–3). The adaptive response is orig- result, inhibits p53 degradation by blocking both p53-MDM2 nuclear inally observed in cells pre-exposed to a low or very low dose of IR export (35, 36) and p53 ubiquitination (37–39). As such, the balance (4–7). Some tumor cells treated in vitro with relative high doses of IR, of MDM2 and p14ARF protein levels may play a critical role in the e.g., fractionated IR (FIR), also demonstrate a transient radioresis- regulation of p53 causing cell phenotypic alterations after chronic IR. tance that appears to be transient during growth (8–10). The molec- By pairwise analysis of radioresistant MCFϩFIR cells that were ular mechanism underlying this kind of stress response that may be derived from chronic FIR with sham-FIR control MCF-7 cells, the causally related to tumor response to radiotherapy has not been present study was designed to determine whether p53 is regulated elucidated. Although many stress signaling genes are activated by IR because of the alteration of p53-regulating proteins. Results of lucif- (11), only a small fraction of genes, i.e., elements of cell cycle control, erase reporter and DNA microarray analysis demonstrated that p53 apoptosis/antiapoptosis, and DNA repair (12, 13) are believed to play was activated and that transcripts of p21, MDM2, and p14ARF, as a key role in the signaling network of IR-induced phenotypic changes. well as a group of stress-responsive genes, were up-regulated by Using cell lines derived from chronic exposure of FIR we have chronic exposure to IR. However, time course analysis showed dif- reported that stress-responsive transcription factors nuclear factor ferent protein levels induced by IR in sham-FIR control versus (NF)-␬B, p53, and AP-1 are activated by FIR (10, 14, 15). The MCFϩFIR cells. No difference was detected in p21 induction. mechanism causing the activation of these transcription factors by MDM2 was inhibited and, correspondingly, MDM2 inhibitor chronic radiation have not been identified. p14ARF was prominently activated in MCFϩFIR cells. Compared Two major phenotypic alterations, growth arrest and apoptosis, are with sham-FIR control cells, MCFϩFIR cells showed an increased linked with p53 activation after a variety of stress conditions including level of phosphorylated p53 (Thr 55) in the cytoplasm and the nucleus IR (16, 17). p53-induced growth arrest is believed due to the delay at of MCFϩFIR, indicating that MDM2 inhibition and p14ARF activa- G1-S or G2-M boundaries that is required for apoptosis (18–21). More tion contribute to a high responsiveness of p53 in chronic IR-derived breast cancer cells. Received 4/10/03; revised 9/19/03; accepted 10/20/03. 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 MATERIALS AND METHODS 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Jian Jian Li, Halper South Building, City of Hope National ؉ Medical Center, 1500 Duarte Road, Duarte, CA 91010. Phone: (626) 301-8355; Fax: Analysis of IR-Derived MCF FIR Cells. MCF-7 cells (starting at pas- (626) 301-8892; E-mail: [email protected]. sage 168) were purchased from American Type Culture Collection. MCFϩFIR 221 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2004 American Association for Cancer Research. REGULATION OF p53 IN CHRONIC RADIATION-TREATED CELLS cells were obtained from MCF-7 cells by exposure to FIR with a total dose of integrity on an agarose gel, RNA was digested using RNase-free DNaseI for 20 60 Gy ␥-irradiation. Radiation was delivered at room temperature at a rate of min, was extracted with phenol-chloroform, and then was precipitated with 2.5 46 cGy/min (Theratron-80 S/N 140 Co-60 Unit; Atomic Energy of Canada volume of ethanol. Polyadenylic acid ϩ RNA was isolated using the Oligotex Limited). MCF-7 cells, treated with the same procedure but without FIR, were RNA kit (Qiagen Inc., Valencia, CA). Gene expression was analyzed using the maintained as sham-FIR control cells. Both sham-FIR control and MCFϩFIR Atlas Human Cancer cDNA Expression Array filter (588–1176 genes) from cells were cultured in DMEM, and experiments were performed with the Clontech (Clontech Laboratories, Inc., Palo Alto, CA). For hybridization, 1 ␮g MCFϩFIR cells within 7–10 passages after the termination of FIR. of polyadenylic acid ϩ RNA was transcribed with nucleotides containing Determining IR-Induced Apoptosis. The terminal deoxynucleotidyl- [␣-32P]dATP, and the labeled cDNA was purified, denatured, and added to 5 transferase-mediated UTP end labeling (TUNEL) method was used to ml of ExpressHyb Hybridization solution (Clontech). The final probe with a detect apoptotic cells. Briefly, cells with or without 5 Gy of IR were concentration of 1 ϫ 106 cpm/ml was freshly applied to the array membrane incubated at 37°C, and, at times after IR, both attached and floating cells that was prehybridized in the ExpressHyb Hybridization solution (Clontech) were harvested and mixed. After washing with PBS, cells were dropped on for 30 min. Hybridization was allowed to proceed overnight at 68°C in a roller to slide glasses and were dried with cold air. All of the cells were fixed for bottle. 30 min at room temperature with 4% paraformaldehyde PBS. The slides The filters were then stringently washed with agitation for 20 min in 200 ml were coded and evaluated using the ϫ40 objective with a photomicrogra- of prewarmed (68°C) solution 1 (2ϫ SSC, 1% SDS) and twice with solution phy rectangle. Ten fields were evaluated in each slide to obtain a mean 2 (0.1ϫ SSC, 0.5% SDS) before being exposed to X-ray film overnight at number for the presence of up to 2000 positive cells for each experimental Ϫ80°C.
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