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

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[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. The filters were also exposed to a Phosphor Screen overnight and data point. The fraction of apoptotic cells was expressed as the percentage scanned using a Storm 840 PhosphorImager (Molecular Dynamics, Inc., of the total number of cells and was determined in three independent Sunnyvale, CA), and signals of paired genes were quantified by ImageQuant experiments for each time point. software. Results represent the quantitation of one hybridization with two sets Cell Cycle Distribution after IR. Sham-FIR control and MCFϩFIR cells of fluorescence-labeled RNA probes, and changes less than 2-fold were not were growing in complete medium and were harvested at different times after listed. exposure to a singe dose of 5 Gy of IR and were fixed in 75% ethanol. The Western Blot. Ten to 20 ␮g of cytoplasmic or nuclear proteins were mixed fixed cells were collected and resuspended in 1 ml of staining buffer, contain- with 50 ␮l of loading buffer, heated at 70°C for 10 min, size-separated in ing 0.1% sodium, 50 ␮g/ml propidium iodide, 0.1% Triton X-100, and were 12.5% acrylamide SDS-PAGE, and transferred to nitrocellulose membranes further incubated overnight at 4°C in the dark. Flow cytometry was performed (Bio-Rad, Hercules, CA). The membranes were then blocked at room temper- to detect propidium iodide-stained nucleus with a the system of FACScan Plus ature for2hinblocking solution (Pierce Co., Rockford, IL), washed with (Becton Dickinson, San Jose, CA), and the data from 1 ϫ 104 cells were 0.01% Tween PBS, and incubated overnight at 4°C with antibodies to MDM2 collected and analyzed using Multicycle software. (sc-5304), p21 (sc-817), and p14ARF (sc-8340) purchased from Santa Cruz Reporter Transfection and Luciferase Assay. Sham-FIR control and Biotechnology (Santa Cruz, CA). The blots were then incubated with horse- MCFϩFIR cells were grown in 12-well cell culture flasks until they reached radish peroxidase-conjugated secondary antibody at a dilution of 1:3000. 70% confluence. For gene transfection, 0.3 ␮g of p53-controlled luciferase Protein bands were visualized using the ECL Plus detection system (Amer- reporters (40) and 0.2 ␮gof␤-galactosidase reporters were cotransfected using sham Life Science, Arlington Heights, IL). LipofectAMINE-plus Reagent (Life Technologies, Inc., Gaithersburg, MD). Both MCFϩFIR and sham-FIR control MCF-7 cells were transfected for 3 h RESULTS and were recovered in complete medium for 6 h. The luciferase activity was measured at different times after radiation with a single dose 5 Gy. For the FIR-Induced Radioresistance and Cell Cycle Distribution. As control of reporter transfection efficiency, an aliquot of the same cell lysates we have demonstrated previously, MCF-7 cells derived from chronic was used for the measurement of ␤-galactosidase activity (␤-galactosidase IR MCFϩFIR showed an increased tolerance to IR-induced cytotox- Enzyme Assay system; Promega Inc. Madison, WI), and the luciferase activity icity (15). To determine the apoptotic response of MCFϩFIR cells, ␤ was normalized to -galactosidase activity. MCFϩFIR and sham-FIR control cells were irradiated with a single Purification of Cytoplasmic and Nuclear Proteins. Cytoplasmic and dose of 5 Gy, and apoptotic cells were calculated at different times nuclear proteins were extracted as described previously (41). Briefly, sham- FIR control MCF-7 and MCFϩFIR cells were exposed to a single dose of 5 Gy after IR. of radiation, collected with trypsin, and washed three times with PBS. The cell Although no obvious change was found in basal level of apoptotic pellets were then resuspended in 1 ml of cell lysis buffer containing: 50 mM KCl, 0.5% NP40, 25 mM HEPES (pH 7.8), 32 mM phenylmethylsulfonyl fluoride, 2 ␮g/ml leupeptin, 4 ␮g/ml aprotinin, and 100 ␮M DTT. The lysis mixture was then centrifuged at 4°C for 1 min. The supernatant was collected as cytoplasmic protein, and the pellets were washed once with 0.5 ml of washing buffer by centrifuging at 4°C for 1 min. Cell nuclei were then resuspended in 100 ␮l of nuclear protein extraction buffer containing 500 mM KCl, 25 mM HEPES, and 10% glycerol and were rocked at 4°C for 15 min, and supernatants were saved as nuclear proteins. Gel Shift Analysis. One to three ␮g of nuclear proteins were incubated on ice for 10 min in a total of 20 ␮l of DNA binding buffer containing binding buffer containing: 10 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 5% glycerol, and 50 ␮g/ml polydeoxyinosinnic deoxycytidylic acid strand DNA; they were then incubated for 20 min at room temperature with 5ϫ 104 cpm 32P-labeled p53 oligonucleotides probe (5ЈTAGGCATGTCTAG- GCATGTCTAAGCT-3Ј). The DNA-protein complexes were resolved on 10% native PAGE and visualized in X-ray film. For supershift assay, nuclear extracts were incubated with antibodies at room temperature for 20 min before the addition of the radioactive-labeled DNA oligonucleotides. Fig. 1. Resistance to ionizing radiation (IR)-induced apoptosis. MCF-7 and RNA Purification and DNA Microarray Analysis. RNA preparation and MCFϩfractionated IR (FIR) cells were irradiated with 5 Gy of IR, and both attached and DNA microarray analysis followed the protocol as described earlier (15). floating cells were collected and stained by terminal deoxynucleotidyltransferase-medi- ϩ ated UTP end labeling (TUNEL) at indicated times after IR. Apoptotic cells were Briefly, total RNA was extracted from sham-FIR control and MCF FIR cells calculated with positively stained nuclei collected from three independent experiments .(P Ͻ 0.01; n ϭ 6000 cells/group ,ءء) using TRIzol Reagent (Life Technologies, Inc.). After confirmation of the 222

Table 1 Cell cycle distribution of sham-fractionated ionizing radiation (FIR) control MCF-7 and MCF ϩ FIR cells Sham-FIR control MCF-7 and MCF ϩ FIR cells were treated with (ϩ) or without (Ϫ) a single dose of 5 Gy of IR. Cells were collected with trypsin at indicated times after IR (in hours), fixed in 85% ethanol, and stained with propidium iodide (PI). PI-stained nuclear DNA content, indicating the distribution of cells in G0-G1, S phase, and G2, was determined with FACScan Plus flowcytometer (Becton Dickinson), and the results of 1 ϫ 104 counted cells were analyzed using Multicycle software. No. of cells (%)

G0-G1 S phase G2-M Cell Lines 5 Gy 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h MCF-7 Ϫ 67.78 66.89 68.44 26.11 30.37 27.12 3.22 2.73 2.45 MCF-7 ϩ 69.89 70.79, 67.24 19.22 18.95 17.89 8.34 9.24 8.56 MCF ϩ FIR Ϫ 62.01 66.01 63.43 28.99 30.55 30.37 3.22 3.43 3.28 MCF ϩ FIR ϩ 67.33 69.15 67.88 24.55 26.2 23.66a 3.89 4.63 4.24a a P Ͻ 0.01 compared with the value of sham-FIR control cells as marked in bold. cells (zero time in Fig. 1), IR induced ϳ50% less of apoptotic cells in MCFϩFIR cells compared with sham-FIR control cells (24 h and 48 h after IR; Fig. 1). Cell cycle distribution illustrated in Table 1 shows that no difference was induced by IR in G0-G1 phases, whereas, compared with control MCF-7 cells, more MCFϩFIR cells were found detained in S phase with less cells in G2-M phase. These results suggest that regulation of cell cycle induced by IR stress is altered in chronic IR-derived MCFϩFIR cells. p53 Activation in MCF؉FIR Cells. To determine p53 activation in the IR-derived tumor cells, the basal and 5-Gy-IR-induced p53 activity was measured in sham-FIR control MCF-7 and MCFϩFIR cells. Compared with sham-FIR control, the basal level of p53- dependent luciferase activity was moderately elevated (ϳ2-fold; data not shown) in MCFϩFIR cells. In contract, p53 luciferase activity was strikingly induced in MCFϩFIR cells after 5 Gy of IR (6.3-fold and 7.9-fold at 8 h and 20 h, respectively, in MCFϩFIR Fig. 3. p53 and phosphorylated p53 [P-p53 (Thr 55)] after a single dose of 5 Gy. A, versus 2.0-fold and 3.3-fold at 8 h and 20 h, respectively, in the Western blotting was performed with the indicated antibody of p53 (sc-4246) or phos- sham-FIR control cells; Fig. 2A). The basal DNA binding activity phorylated p53 [P-p53 (Thr 55), sc-12904-R] and 20 ␮g of cytoplasmic or nuclear ϳ proteins isolated from sham-fractionated ionizing radiation (FIR) control MCF-7 and of p53 detected by gel shift assay was increased ( 10-fold) in ϩ ϩ MCF FIR cells with or without 5 Gy of irradiation. B, relative levels of cytoplasmic and MCF FIR cells, and little increase was induced after IR (Fig. 2, nuclear p53 proteins estimated by densitometric analysis (representative of three experi- Lanes 6–8). The sham-FIR control MCF-7 cells showed a low ments). basal DNA binding but predominant induction by IR (ϳ12.3-fold at 24 h; Fig. 2B, Lane 4). These results demonstrate that DNA binding and p53-controlled luciferase transcription are differently regulated in IR-derived MCFϩFIR cells. Increased Phosphorylation and Nuclear Distribution of p53 in MCF؉FIR Cells. Western blot was applied to determine whether increased DNA binding activity of p53 is caused by phosphorylation and protein accumulation. The basal and 5-Gy-IR-induced p53 and Thr 55 phosphorylation was accessed in sham-FIR control MCF-7 and IR-derived MCFϩFIR cells. Total p53 in cytoplasm showed little difference except a slight increase in 5-Gy-treated MCFϩFIR cells (Fig. 3A). In contrast, the basal and IR-induced p53 (Thr 55) was enhanced in both the cytoplasm and the nucleus of MCFϩFIR cells. Interestingly, both total and phosphorylated p53 was slightly reduced in the control MCF-7 cells, which is accompanied by the increased DNA-binding of p53 (Fig. 2), sug- gesting an enhanced p53 degradation in control MCF-7 cells. This inhibitory function appears to be down-regulated in IR-derived MCFϩFIR cells. Fig. 2. p53 activation in MCFϩfractionated ionizing radiation (FIR) cells. A, increased p53-Responsive Genes Detected by DNA Microarray Analysis. p53-dependent luciferase transactivation. p53-luciferase and ␤-galactosidase reporter ϩ DNA microarray analysis was then applied to detect effector genes plasmids were cotransfected into control MCF-7 and MCF FIR cells using Lipo- ϩ fectAMINE and Plus Reagent Kit. Cells were irradiated 3 h after DNA transfection by 5 differently regulated in MCF FIR versus sham-FIR control cells. Gy of IR. p53 transactivation was determined by luciferase activities measured at the We have reported a cluster of effector genes activated in the ␤ indicated times after IR, and luciferase activity was normalized to -galactosidase activity radioresistance induced by expression of MnSOD and/or FIR (15). (mean and SE of five experiments; some error limits are hidden in the symbols). B, p53/DNA binding activity after a single dose of 5 Gy of IR. Gel shift analysis was The present study shows gene expression levels in MCFϩFIR performed with 32P-labeled p53 consensus oligonucleotide and 4 ␮g of nuclear protein versus the sham-FIR control cells using 588-1176 grouped human purified from sham-FIR control MCF-7 and MCFϩFIR cells. Lanes 0, without 5 Gy of IR. p53/DNA binding activity was estimated by densitometry (representative of three exper- gene fragments (Clontech Atlas Human Cancer cDNA). Each gene iments) was pair-wise analyzed by comparison of the expression level from 223

Table 2 Down-regulated genes in comparison of MCF ϩ fractionated ionizing radiation (FIR) versus sham-FIR control MCF-7 cellsa GenBank no. Gene name Gene function n-foldb change Function ϭ apoptosis 1. U69611 TNF-␣ converting enzyme Apoptosis 4.9 2. Y09392 WSL-LR (Apo-3) Apoptosis 3.9 3. X60188 Tumor necrosis factor receptor 2 precursor Apoptosis 2.8 Function ϭ other 4. M16961 ␣-2-HS-glycoprotein precursor Insulin tyrosine kinase inhibitor 4.9 5. L13689 DNA-binding protein BMI-1 Signal transduction 4.2 6. M14091 Thyroxin-binding globulin precursor TBG function 3.9 7. M32977 Vascular endothelial growth factor (vpf) Growth factor 3.6 8. D12763 ST2 protein precursor Cytokine 3.6 9. M24283 Intercellular adhesion molecule-1 precursor Cell adhesion 3.5 10. X69550 Rho GDP-dissociation inhibitor-1 GTPase regulator 3.2 11. M10901 Glucocorticoid receptor ␣ Transcription factor 3.1 12. M59911 Integrin ␣-3 Adhesion molecules 2.2 13. M96995 Growth factor receptor-bound protein 2 T4-binding globulin 2.1 14. Q00421 GA Binding protein ␤-2 chain Transcription factor 2.0 a Results represent the quantitation of two hybridizations of two sets of fluorescence-labeled RNA probes, not including values with changes of less than 2-fold. b MCF ϩ FIR profiles were obtained by normalizing gene expression levels of MCF ϩ FIR population to the level of sham-FIR treated MCF-7 cells.

MCFϩFIR to the level of sham-FIR control MCF-7 cells. Genes INKA-ARF (3.9-fold), IAP3 (4.1-fold), Cyclin B1 (3.3-fold), Cy- found to be down-regulated (Ͻ2-fold decrease compared with clin D1 (3.4-fold), and others were up-regulated in MCFϩFIR MCF-7; Table 2) or up-regulated (Ͼ2-fold increase compared cells (marked in the list of Table 3). p14ARF, an alternative gene with MCF-7) were grouped by their functions and were ranked product of INKARF (39) has been indicated to act as a negative with gene expression levels (Table 3). A group of signaling ele- control of MDM2 and thus controls MDM2-mediated p53 regu- ments associated with p53 regulation and signaling apoptosis lation. and/or antiapoptosis including MDM2 (3.8-fold), p21 (4.6-fold), Because p21, MDM2, and p14ARF are closely linked with p53 reg-

Table 3 Up-regulated genes in comparison of MCF ϩ fractionated ionizing radiation (FIR) versus sham-FIR control MCF-7 cellsa GenBank no. Gene name Function n-foldb change Function ϭ apoptosis 1. L25081 Transforming protein RHOC Apoptosis, proliferation, carcinogenesis 5.8 2. V00568 MYC proto-oncogene protein Antiapoptosis 5.5 3. M86400 14-3-3 ␨ Antiapoptosis 5.1 4. U09579 p21(waf1, CDKN1A) Apoptosis and cell cycle regulator 4.6c 5. S78085 PDCD2 Apoptosis 4.6 6. U45880 IAP3 Antiapoptosis 4.1 7. Z12020 MDM2 Apoptosis-associated protein 3.8 8. L27211 INK4A-ARF Cyclin-dependent kinase inhibitor 3.9 9. S83171 BCL-2 binding protein-1 (BAG-1) Antiapoptosis 2.9 10. U29680 BCL2-related protein A1 Bcl family protein 3.2 11. M14745 BCL-2-␣ Antiapoptosis 3.1 12. Q99537 BRCA1, RHO7, AND VATI Apoptosis 2.2 13. U76248 HSIAH2 Apoptosis-associated protein 2.1 14. AI376462 Lupus KU autoantigen protein P86 Apoptosis, radioresistance 2.0 Function ϭ cell cycle control 15. S40706 GADD 153 (Growth arrest and DNA-damage-inducible protein) DNA damage response Cell cycle control 5.4 16. U54777 DNA mismatch repair protein MSH6 DNA repair 4.7 17. X00588 EGFR Cell proliferation 4.5 18. U04045 DNA mismatch repair protein MSH2 DNA repair 3.8 19. X12530 B-Lymphocyte antigen CD20 Cell cycle antigen 3.5 20. X59798 Cyclin D1 G1-S phase cell cycle adjustment 3.4 21. M25753 Cyclin B1 G2-mitotic cell cycle adjustment 3.3 22. X51688 Cyclin A Cell cycle control 3.3 Function ϭ other 23. U57342 Myeloid leukemia factor 2 (MLF2) Cell differentiation 8.9 24. M55422 Zinc finger protein Nuclear receptors 8.3 25. Y00371 Heat-shock cognate 71kD protein Stress response 8.3 26. AF026816 Putative oncogene protein Oncogene 6.9 27. M65199 Endothelin 2 Growth factor 6.8 28. M27396 Asparagine synthetase Cell proliferation 6.3 29. U43901 Laminin, 37KD receptor Cell adhesion 5.6 30. S72008 CDC10 protein homolog Growth regulator 4.4 31. M22489 Bone morphogenetic protein 2A Growth factor 4.3 32. D13748 Eukaryotic initiation factor 4A-I Cell proliferation 4.1 33. M63488 Single stranded DNA-binding protein DNA replication 4.0 34. M38690 CD9 Cell adhesion 3.5 35. M14091 Thyroxin-binding globulin precursor Stress response 3.4 36. L07594 TGF-␤ receptor Type III Precursor Cell proliferation, differentiation 3.3 37. L38503 Glutathione S-transferase ␪ 2 Stress response 3.2 38. Q00421 GA Binding protein ␤-2 chain Transcription factor 3.2 39. X60221 ATP Synthase B chain, mitochondrial precursor ATP synthesis 3.2 40. U72661 Ninjurin-1 Cell adhesion 2.0 a Results represent the quantitation of two hybridizations of two sets of fluorescence-labeled RNA probes, not including values with changes of less than 2-fold. b MCF ϩ FIR profiles were obtained by normalizing gene expression levels of MCF ϩ FIR population to the level of sham-FIR treated MCF-7 cells. c Underlining indicates the genes associated with p53 function and the time-course of protein expression (numbers 4, 7, and 8) was analyzed in MCF ϩ FIR versus control MCF-7 cells. 224

Transcriptional and Protein Levels of MDM2 Inhibitor p14ARF Were Increased in MCF؉FIR Cells. Two proteins have been reported by the splicing of the mRNA of INKARF: p16ARF (interaction with Rb protein), and p14ARF (interaction with MDM2; Ref. 38). Most recently, p14ARF has been linked to MDM2 protein degradation (38, 42). INKARF transcripts were up-regulated 3.8-fold in MCFϩFIR cells (Table 3). The increased p14ARF that reduces p53 degradation by MDM2, may contribute to the high p53 level of protein in MCFϩFIR cells. p14ARF mRNA and protein levels were analyzed with or without 5 Gy of IR (Fig. 6). Using published primers of p14ARF (43), reverse transcription-PCR showed that basal p14ARF transcripts were very Fig. 4. No difference in ionizing radiation (IR)-induced p21 protein expression and low but were enhanced by 5 Gy of IR (3-fold) in sham-FIR control nuclear distribution. A, Western blotting was performed with 20 ␮g of cytoplasmic or ϩ cells (Fig. 6A, Lanes 1 and 2). However, basal p14ARF transcripts nuclear proteins isolated from sham-fractionated IR (FIR) control MCF-7 and MCF FIR ϳ cells at times indicated after 5 Gy of irradiation. The nonspecific bands (NS) were included were much more highly increased ( 9-fold) and were maintained at to show equal loading of proteins (representative of three experiments). B, relative levels a similar level in MCFϩFIR cells after 5 Gy of IR (Fig. 6A, Lanes 3 of cytoplasmic and nuclear p21 proteins estimated by the ratio of p21:NS bands measured and 4). Consistent with the increased transcripts, p14ARF proteins with densitometry. that were detected in cytoplasmic and nuclear extracts were 6–8-fold higher in MCFϩFIR cells compared with the levels of sham-FIR control cells (Fig. 6, B and C). These results suggest that the expres- ulation, we further analyzed the time course changes of the protein levels sion of p14ARF and the inhibition of MDM2 work together to of p21, MDM2, and p14ARF and their nuclear distribution after IR. maintain p53 protein in the nucleus causing a high responsiveness of ؉ p21 Induced by IR in Both Sham-FIR Control and MCF FIR p53 in chronic IR-derived radioresistant cells. Cells. To confirm the protein levels of p21 expression detected by microarray analysis, Western blot was performed using cytoplasmic DISCUSSION and nuclear proteins of sham-FIR control and MCFϩFIR cells with or without 5 Gy of IR (Fig. 4). Although the basal p21 protein expression The present findings demonstrate that p53 is actively involved in was slightly increased (in agreement with up-regulated gene tran- human breast carcinoma MCFϩFIR cells that showed a transient scripts shown in Table 3) in the cytoplasm and nucleus of MCFϩFIR radioresistance after chronic exposure to IR. p53 transcriptional ac- cells (2-fold in cytoplasmic and 3-fold in nuclear proteins), no differ- tivity was increased with little recruitment of p53 in DNA binding; ence was detected in p21 induction and nuclear translocation after 5 Gy of IR (Fig. 4). These results suggest that the p21 expression level may not act as a rate-limiting factor in signaling IR-derived radioresistance. MDM2 Induction and Nuclear Distribution Was Inhibited in MCF؉FIR Cells. Our microarray data showed an elevated MDM2 mRNA level in MCFϩFIR cells (3.8-fold; Table 3). To get into the inside mechanisms underlying p53 activation in MCFϩFIR cells, distribution of cytoplasmic and nuclear MDM2 as well as MDM2 in p53 DNA-binding complexes were analyzed. The basal MDM2 was detectable only in MCFϩFIR cells but not in the sham-FIR control cells (Fig. 5A, Lane 5 normalized to the background of Lane 1). However, surprisingly, MDM2 immunoreactive protein was not induced during a time period of 3–24 h after IR (Fig. 5A, Lanes 5–8). In contrast, the cytoplasmic MDM2 was markedly enhanced (ϳ7-fold at 3 h) in sham-FIR control cells (Fig. 5A, Lanes 1–4) and detected in the nuclear protein purified from sham-FIR control cells treated with IR. However, no MDM2 nuclear distribution was detected in ϩ MCF FIR cells with or without IR (Fig. 5B). Fig. 5. MDM2 expression and nuclear translocation were activated in sham-fraction- We then asked the question of whether the nuclear-distributed ated ionizing radiation (FIR) control MCF-7 but not in MCFϩFIR cells. A, cytoplasmic MDM2 combines to p53/DNA complexes leading to a feed-back MDM2 was induced by IR in sham-FIR control MCF-7 but not in MCFϩFIR cells. Western blot was performed with 20 ␮g of protein from control MCF-7 and MCFϩFIR control of p53 activation. Supporting this concept, supershifting anal- cells at the indicated time intervals after 5 Gy of irradiation. MDM2 was visualized with ysis with antibody to MDM2 demonstrated that MDM2 was present in MDM2 antibody, and expression levels were estimated by the ratio of MDM2:nonspecific bands (NS) measured with densitometry (lower panel, representative of three experi- the p53/DNA complex of the sham-FIR control cells 24 h after 5 Gy ments). B, MDM2 was detected in the nucleus of sham-FIR control MCF-7 but not in of IR (Fig. 5C, Lanes 1 and 2), and MDM2 was not detected in the MCFϩFIR cells. Nuclear MDM2 was measured by Western blot with 20 ␮g of nuclear p53/DNA complex of MCFϩFIR cells with or without 5 Gy of IR proteins purified from sham-FIR control MCF-7 or MCFϩFIR cells 24 h after treatment with (ϩ) or without (Ϫ) 5 Gy of IR. The nuclear proteins were separated on 12.5% (Fig. 5C, Lanes 3 and 4). These results demonstrate that MDM2/p53 acrylamide gel, and MDM2 was detected by MDM2 antibody. Nonspecific bands (NSs) binding occurs in the nucleus of control MCF-7 cells, indicating a were indicated as loading controls (representative of three experiments). C, MDM2 was feed-back control of p53 by MDM2 in IR-induced stress response. detected in the p53/DNA complex of sham-FIR control MCF-7 but absent in the p53/DNA complex of MCFϩFIR cells. Supershifting of MDM2 was performed with 32P-labeled This regulation mechanism appears to be down-regulated in chronic p53 consensus oligonucleotide and 4 ␮g of nuclear proteins purified from sham-FIR IR-derived MCFϩFIR cells. Lack of phosphorylated p53 (Thr 55) in control MCF-7 and MCFϩFIR cells 24 h with (ϩ) or without (Ϫ)5GyofIR.The ϩ protein/DNA complex was then separated by gel electrophoresis. Arrows, p53/DNA control cells, but increased in MCF FIR cells (Fig. 3), laid a support complex and the shifted MDM2 proteins. Lower panel, the estimated ratio of MDM2 for this hypothesis. super-shifted bands:the level of p53/DNA complex (representative of three experiments). 225

MDM2 was detected in the p53/DNA binding complex of sham-FIR control but not IR-derived MCFϩFIR cells (Fig. 5, B and C). On the basis of the interaction of MDM2 with p53, our results suggest that MDM2 functions to inhibit p53 in cells with a normal status of p53, and this interaction is inhibited in chronic IR-derived cells that showed a transient radioresistance. Interaction of MDM2 with p53

NH2-terminal region accelerates p53 degradation and inhibits the ability of p53-mediated gene transcription (50). In addition, the sequence required for MDM2-mediated p53 nuclear export has also been identified (34, 50). Therefore, MDM2 accumulated in the control sham-FIR cells after IR appears to be required for a feed-back control of p53 activation because p53/DNA binding was increased with a relative low enhancement of p53-controlled luciferase activity. Fur- ther evidence is provided by the fact that MDM2 was directly detected in p53/DNA complex in control MCF-7 but not in IR-derived MCFϩFIR cells (Figs. 5, B and C). Down-regulation of the p53 inhibitory element may be a critical adjustment to preserve p53 proteins in cells adapted to chronic IR. Mechanisms underlying MDM2 inhibition in IR-derived cells need to be identified.

Fig. 6. p14ARF mRNA and protein levels were increased in MCFϩfractionated An important finding in the present microarray and protein expres- ionizing radiation (FIR) cells. A, p14ARF mRNA level was increased in MCFϩFIR cells. sion studies is the activation of p14ARF. In mammalian cells, the p14ARF transcripts were detected by reverse transcription-PCR using the published INK4␣-p14ARF locus has been identified to encode two different cell ␮ p14ARF primers shown in “Materials and Methods” and 5 g of total RNA of sham-FIR ␣ control (C) MCF-7 and MCFϩFIR cells. Gene fragments were enhanced for 25 cycles, cycle inhibitors (p16INK4 and p14ARF) by alternative splicing (51, and relative levels of transcripts were estimated by densitometric analysis and were 52). It has been suggested that ARF family proteins regulate p53 presented as arbitrary units after normalization to the level of GAPDH (representative of activity (36, 53) via a negative regulation by MDM2 (53, 54). three experiments). B, Western blot was performed with p14ARF antibody and 20 ␮gof cytoplasmic or nuclear proteins of control MCF-7 and MCFϩFIR cells 24 h after 5 Gy p14ARF is also required for MDM2 degradation by small ubiquitin- of IR (representative of three experiments). C, the cytoplasmic and nuclear p14ARF levels like protein (SUMO; Ref. 42). In the present study, we found that were estimated by the ratio of p14ARF:Actin bands measured with densitometry. p14ARF was activated in MCFϩFIR cells that showed down-regulation of MDM2 and up-regulation of p14ARF. As a result, p53 lucif- and p53-modulating protein MDM2 was inhibited and MDM2-inhib- erase transcription was much higher in MCFϩFIR cells than control itory protein p14ARF was activated in MCFϩFIR cells. These results cells after IR. Correspondingly, expression of p14ARF and MDM2 suggest that p53-regulating elements MDM2 and p14ARF play a key was found to be oppositely regulated in sham-FIR control cells after role in the up-regulation of the p53 activity under chronic stresses IR. This difference between control and chronic IR-derived cells with IR. suggests an adjustment of p14ARF and MDM2 proteins, which ap- p53 signaling network has been shown to play a central role in pears to be required for maintaining a relatively high level of p53 maintaining the genomic integrity and protecting cells against IR- responsiveness in IR-derived cells. This adjustment may contribute to induced damage (18, 26, 27). The present results indicate an elevated a quick transactivation of p53 without the recruitment of p53 into the “constitutive” DNA binding and transcriptional activity of p53 in nucleus. IR-derived MCFϩFIR cells that showed an enhanced resistance to Inhibition of p53 has radiosensitized human diploid fibroblasts (55) IR-induced apoptosis. It has been well accepted that activation of p53 and expression of p53 is associated with local failure of neck and head is able to adjust the cell cycle time to allow cells to repair the damaged tumors in radiotherapy (56). Although a direct linking between p53 DNA induced by IR (44). IR-induced cell cycle delay is attributed to activation and radioresistance has not been identified, transcription the activation of p53 (45), which induces cyclin-dependent kinase- factor NF-␬B, evidently related to radioresistance (10, 57, 58), is inhibitory protein p21, which is, in turn, required for specific cell associated with p53 activation. The inhibition of NF-␬B abrogates cycle checkpoints (30, 46). However, p21 showed little response to p53-induced apoptosis (59), and expression of p65, the key subunit of anticancer agent-induced stress in some tumor cells. Loss of p21 NF-␬B, activates p53 gene promoter activity (60). We have reported results in an induction of apoptosis, but no difference was found in the that both p53 and NF-␬B are activated by IR in MCF-7 cells (9), and clonogenic survival (47, 48), in IR-induced S-phase checkpoint (41), NF-␬B activation is causally related to IR-derived radioresistance (10, or in DNA repair responses to UV radiation (21). We report here that, 15). The coordinated modulation of p53 and NF-␬B pathways has although p53 and p21 were activated in IR-derived MCFϩFIR cells, been indicated in IR-induced response in human malignant melanoma no difference was detected in p21 protein and nuclear translocation in cells (61). control and MCFϩFIR cells after IR. In addition, our published When we follow this line of reasoning, p53 appears to participate results have shown that blocking p21 by antisense transfection did not the radioresistance-signaling network leading to the activation of enhance the radiosensitivity of IR-derived MCFϩFIR cells (9). These NF-␬B. In addition, p53 and NF-␬B likely regulate the function of results argue that although p21 transcription can be up-regulated Ku86 a subunit of DNA-dependent protein kinase (DNA-PK) and a because of p53 activation, p21 protein may not function as a rate- key protein in DNA repair (62). Our microarray data showed that, limiting element in chronic IR-induced stress response. compared with sham-FIR control cells, Ku86 transcripts were up- The reported (49) and present data (Table 3; Fig. 5) demonstrate regulated in IR-derived MCFϩFIR cells (Table 3). Because Ku86 and that MDM2 transcripts are increased by IR. To determine MDM2 its isoform KARP-1 can be regulated by p53 (63) and, importantly, response in IR-derived cell, however, we found that MDM2 was DNA-dependent protein kinase is shown to phosphorylate I␬B␣ for inhibited in MCFϩFIR cells after the stress with a single dose of IR. NF-␬B activation (64), NF-␬B can be activated in IR-derived cells by In contrast, MDM2 was induced (ϳ7-fold) by IR (Fig. 5A) and Ku86 as a result of p53 activation. Such a Ku86-mediated connection 226

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Liqun Xia, Aimee Paik and Jian Jian Li

Cancer Res 2004;64:221-228.

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