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[Cancer Research 64, 221-228, January 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

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
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian cells chronically exposed to ionizing radiation (IR) induce stress response with a tolerance to the subsequent cytotoxicity of IR. Although p53 is well documented in IR response, the signaling network causing p53 activation in chronic IR remains to be identified. Using breast carcinoma MCF+FIR cells that showed a transient radioresistance after exposure chronically to fractionated IR (FIR), the present study shows that the basal DNA binding and transcriptional activity of p53 was elevated by FIR. p53-controlled luciferase activity was strikingly induced (~7.9-fold) with little enhancement of p53/DNA binding activity (~1.3-fold). The phosphorylated p53 (Thr 55) was increased in the cytoplasm and nucleus of MCF+FIR but not in the sham-FIR control cells. On the contrary, the sham-FIR control MCF-7 cells showed a low p53 luciferase transcription (~3-fold) but a striking enhancement of p53/DNA binding (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 as a reference demonstrated that the mRNA of p21, MDM2, and p14ARF was up-regulated. Time course Western blot analysis, however, showed no difference in p21 induction. In contrast, MDM2 that was absent in control cells and was predominantly induced by IR was not induced in MCF+FIR cells. In agreement with MDM2 inhibition, MDM2-inhibitory protein p14ARF was increased in MCF+FIR cells. In summary, these results demonstrate that up-regulation of p14ARF paralleled with MDM2 inhibition contributes to p53 accumulation in the nucleus and causes a high responsiveness of p53 in chronic IR-treated breast cancer cells.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian cells treated with ionizing radiation (IR) induces a stress response associated with an enhanced tolerance to the subsequent cytotoxicity of radiation (1, 2, 3) . The adaptive response is originally observed in cells pre-exposed to a low or very low dose of IR (4, 5, 6, 7) . Some tumor cells treated in vitro with relative high doses of IR, e.g., fractionated IR (FIR), also demonstrate a transient radioresistance that appears to be transient during growth (8, 9, 10) . The molecular mechanism underlying this kind of stress response that may be causally related to tumor response to radiotherapy has not been elucidated. Although many stress signaling genes are activated by IR (11) , only a small fraction of genes, i.e., elements of cell cycle control, apoptosis/antiapoptosis, and DNA repair (12 , 13) are believed to play a key role in the signaling network of IR-induced phenotypic changes. Using cell lines derived from chronic exposure of FIR we have reported that stress-responsive transcription factors nuclear factor (NF)-{kappa}B, p53, and AP-1 are activated by FIR (10 , 14 , 15) . The mechanism causing the activation of these transcription factors by chronic radiation have not been identified.

Two major phenotypic alterations, growth arrest and apoptosis, are linked with p53 activation after a variety of stress conditions including IR (16 , 17) . p53-induced growth arrest is believed due to the delay at G1-S or G2-M boundaries that is required for apoptosis (18, 19, 20, 21) . More than 100 genes of human genome have been identified as have binding sites for p53 (22) , and many stress genes activated by IR are a result of p53 activation (23 , 24) . With a link to these IR effector genes, p53 activity has been shown to affect the anticancer efficiency of radiotherapy using FIR (25) . Additional evidence suggest that p53 is very sensitive to IR-induced DNA damages (26) , and DNA strand breaks induced by IR are believed to be the major source that trigger the p53-dependent repair system (27) . However, it is unclear how p53 is regulated in cells that showed a transient tolerance to IR after exposure chronically to IR. This transient radioresistant phenotype in radiation-derived cells (8, 9, 10) presents a tolerance of tumor cells to radiotherapy.

Both MDM2 functioning in p53 protein degradation and p14ARF functioning in MDM2 inhibition may be involved in the metabolic regulation of p53 activity (28 , 29) . IR-induced p53 initiates the signaling process that causes cell cycle arrest, which is accompanied by the ability for DNA repair and apoptosis (30 , 31) . This process can be actively regulated by expression of MDM2. Activation of p53 and MDM2 is found in cells arrested at G1-S phase or G2-M boundaries induced by IR (17) . Interaction of MDM2 with the p53 NH2-terminal region has been shown to accelerate p53 degradation and to inhibit the capacity for p53-mediated gene transcription (28 , 29) . In addition, MDM2 is found actively involved in the process of p53 nuclear exclusion (32) , and the sequence required for MDM2-mediated p53 degradation and nuclear export has been identified (33 , 34) . MDM2 is also shown to be a unique ubiquitin-protein ligase to ubiquitinate and degrade p53 (28 , 29) .

However, the inhibitory effect of MDM2 on p53 can be counteracted by p14ARF, a protein that directly binds to MDM2 and, as a result, inhibits p53 degradation by blocking both p53-MDM2 nuclear export (35 , 36) and p53 ubiquitination (37, 38, 39) . As such, the balance of MDM2 and p14ARF protein levels may play a critical role in the regulation of p53 causing cell phenotypic alterations after chronic IR. By pairwise analysis of radioresistant MCF+FIR cells that were derived from chronic FIR with sham-FIR control MCF-7 cells, the present study was designed to determine whether p53 is regulated because of the alteration of p53-regulating proteins. Results of luciferase reporter and DNA microarray analysis demonstrated that p53 was activated and that transcripts of p21, MDM2, and p14ARF, as well as a group of stress-responsive genes, were up-regulated by chronic exposure to IR. However, time course analysis showed different protein levels induced by IR in sham-FIR control versus MCF+FIR cells. No difference was detected in p21 induction.

MDM2 was inhibited and, correspondingly, MDM2 inhibitor p14ARF was prominently activated in MCF+FIR cells. Compared with sham-FIR control cells, MCF+FIR cells showed an increased level of phosphorylated p53 (Thr 55) in the cytoplasm and the nucleus of MCF+FIR, indicating that MDM2 inhibition and p14ARF activation contribute to a high responsiveness of p53 in chronic IR-derived breast cancer cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Analysis of IR-Derived MCF+FIR Cells.
MCF-7 cells (starting at passage 168) were purchased from American Type Culture Collection. MCF+FIR cells were obtained from MCF-7 cells by exposure to FIR with a total dose of 60 Gy {gamma}-irradiation. Radiation was delivered at room temperature at a rate of 46 cGy/min (Theratron-80 S/N 140 Co-60 Unit; Atomic Energy of Canada Limited). MCF-7 cells, treated with the same procedure but without FIR, were maintained as sham-FIR control cells. Both sham-FIR control and MCF+FIR cells were cultured in DMEM, and experiments were performed with the MCF+FIR cells within 7–10 passages after the termination of FIR.

Determining IR-Induced Apoptosis.
The terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL) method was used to detect apoptotic cells. Briefly, cells with or without 5 Gy of IR were incubated at 37°C, and, at times after IR, both attached and floating cells were harvested and mixed. After washing with PBS, cells were dropped on to slide glasses and were dried with cold air. All of the cells were fixed for 30 min at room temperature with 4% paraformaldehyde PBS. The slides were coded and evaluated using the x40 objective with a photomicrography rectangle. Ten fields were evaluated in each slide to obtain a mean number for the presence of up to 2000 positive cells for each experimental data point. The fraction of apoptotic cells was expressed as the percentage of the total number of cells and was determined in three independent experiments for each time point.

Cell Cycle Distribution after IR.
Sham-FIR control and MCF+FIR cells were growing in complete medium and were harvested at different times after exposure to a singe dose of 5 Gy of IR and were fixed in 75% ethanol. The fixed cells were collected and resuspended in 1 ml of staining buffer, containing 0.1% sodium, 50 µg/ml propidium iodide, 0.1% Triton X-100, and were further incubated overnight at 4°C in the dark. Flow cytometry was performed to detect propidium iodide-stained nucleus with a the system of FACScan Plus (Becton Dickinson, San Jose, CA), and the data from 1 x 104 cells were collected and analyzed using Multicycle software.

Reporter Transfection and Luciferase Assay.
Sham-FIR control and MCF+FIR cells were grown in 12-well cell culture flasks until they reached 70% confluence. For gene transfection, 0.3 µg of p53-controlled luciferase reporters (40) and 0.2 µg of ß-galactosidase reporters were cotransfected using LipofectAMINE-plus Reagent (Life Technologies, Inc., Gaithersburg, MD). Both MCF+FIR and sham-FIR control MCF-7 cells were transfected for 3 h 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 control of reporter transfection efficiency, an aliquot of the same cell lysates was used for the measurement of ß-galactosidase activity (ß-galactosidase Enzyme Assay system; Promega Inc. Madison, WI), and the luciferase activity was normalized to ß-galactosidase activity.

Purification of Cytoplasmic and Nuclear Proteins.
Cytoplasmic and nuclear proteins were extracted as described previously (41) . Briefly, sham-FIR control MCF-7 and MCF+FIR cells were exposed to a single dose of 5 Gy of radiation, collected with trypsin, and washed three times with PBS. The cell 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 µg/ml leupeptin, 4 µg/ml aprotinin, and 100 µM 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 µl 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 µg of nuclear proteins were incubated on ice for 10 min in a total of 20 µl 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 µg/ml polydeoxyinosinnic deoxycytidylic acid strand DNA; they were then incubated for 20 min at room temperature with 5x 104 cpm 32P-labeled p53 oligonucleotides probe (5'TAGGCATGTCTAGGCATGTCTAAGCT-3'). 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.

RNA Purification and DNA Microarray Analysis.
RNA preparation and DNA microarray analysis followed the protocol as described earlier (15) . Briefly, total RNA was extracted from sham-FIR control and MCF+FIR cells using TRIzol Reagent (Life Technologies, Inc.). After confirmation of the integrity on an agarose gel, RNA was digested using RNase-free DNaseI for 20 min, was extracted with phenol-chloroform, and then was precipitated with 2.5 volume of ethanol. Polyadenylic acid + RNA was isolated using the Oligotex RNA kit (Qiagen Inc., Valencia, CA). Gene expression was analyzed using the Atlas Human Cancer cDNA Expression Array filter (588–1176 genes) from Clontech (Clontech Laboratories, Inc., Palo Alto, CA). For hybridization, 1 µg of polyadenylic acid + RNA was transcribed with nucleotides containing [{alpha}-32P]dATP, and the labeled cDNA was purified, denatured, and added to 5 ml of ExpressHyb Hybridization solution (Clontech). The final probe with a concentration of 1 x 106 cpm/ml was freshly applied to the array membrane that was prehybridized in the ExpressHyb Hybridization solution (Clontech) for 30 min. Hybridization was allowed to proceed overnight at 68°C in a roller bottle.

The filters were then stringently washed with agitation for 20 min in 200 ml of prewarmed (68°C) solution 1 (2x SSC, 1% SDS) and twice with solution 2 (0.1x SSC, 0.5% SDS) before being exposed to X-ray film overnight at -80°C. The filters were also exposed to a Phosphor Screen overnight and scanned using a Storm 840 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA), and signals of paired genes were quantified by ImageQuant software. Results represent the quantitation of one hybridization with two sets of fluorescence-labeled RNA probes, and changes less than 2-fold were not listed.

Western Blot.
Ten to 20 µg of cytoplasmic or nuclear proteins were mixed with 50 µl of loading buffer, heated at 70°C for 10 min, size-separated in 12.5% acrylamide SDS-PAGE, and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). The membranes were then blocked at room temperature for 2 h in blocking solution (Pierce Co., Rockford, IL), washed with 0.01% Tween PBS, and incubated overnight at 4°C with antibodies to MDM2 (sc-5304), p21 (sc-817), and p14ARF (sc-8340) purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The blots were then incubated with horseradish peroxidase-conjugated secondary antibody at a dilution of 1:3000. Protein bands were visualized using the ECL Plus detection system (Amersham Life Science, Arlington Heights, IL).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
FIR-Induced Radioresistance and Cell Cycle Distribution.
As we have demonstrated previously, MCF-7 cells derived from chronic IR MCF+FIR showed an increased tolerance to IR-induced cytotoxicity (15) . To determine the apoptotic response of MCF+FIR cells, MCF+FIR and sham-FIR control cells were irradiated with a single dose of 5 Gy, and apoptotic cells were calculated at different times after IR.

Although no obvious change was found in basal level of apoptotic cells (zero time in Fig. 1Citation ), IR induced ~50% less of apoptotic cells in MCF+FIR cells compared with sham-FIR control cells (24 h and 48 h after IR; Fig. 1Citation ). Cell cycle distribution illustrated in Table 1Citation shows that no difference was induced by IR in G0-G1 phases, whereas, compared with control MCF-7 cells, more MCF+FIR 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 MCF+FIR cells.



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  		Fig. 1. Resistance to ionizing radiation (IR)-induced apoptosis. MCF-7 and MCF+fractionated IR (FIR) cells were irradiated with 5 Gy of IR, and both attached and floating cells were collected and stained by terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL) at indicated times after IR. Apoptotic cells were calculated with positively stained nuclei collected from three independent experiments (**, P < 0.01; n = 6000 cells/group).

 

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  		Table 1 Cell cycle distribution of sham-fractionated ionizing radiation (FIR) control MCF-7 and MCF + FIR cells

Sham-FIR control MCF-7 and MCF + FIR cells were treated with (+) or without (-) 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 x 104 counted cells were analyzed using Multicycle software.

 
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 MCF+FIR cells.

Compared with sham-FIR control, the basal level of p53-dependent luciferase activity was moderately elevated (~2-fold; data not shown) in MCF+FIR cells. In contract, p53 luciferase activity was strikingly induced in MCF+FIR cells after 5 Gy of IR (6.3-fold and 7.9-fold at 8 h and 20 h, respectively, in MCF+FIR versus 2.0-fold and 3.3-fold at 8 h and 20 h, respectively, in the sham-FIR control cells; Fig. 2ACitation ). The basal DNA binding activity of p53 detected by gel shift assay was increased (~10-fold) in MCF+FIR cells, and little increase was induced after IR (Fig. 2Citation , Lanes 6–8). The sham-FIR control MCF-7 cells showed a low basal DNA binding but predominant induction by IR (~12.3-fold at 24 h; Fig. 2Citation B, Lane 4). These results demonstrate that DNA binding and p53-controlled luciferase transcription are differently regulated in IR-derived MCF+FIR cells.



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  		Fig. 2. p53 activation in MCF+fractionated ionizing radiation (FIR) cells. A, increased p53-dependent luciferase transactivation. p53-luciferase and ß-galactosidase reporter plasmids were cotransfected into control MCF-7 and MCF+FIR cells using LipofectAMINE and Plus Reagent Kit. Cells were irradiated 3 h after DNA transfection by 5 Gy of IR. p53 transactivation was determined by luciferase activities measured at the indicated times after IR, and luciferase activity was normalized to ß-galactosidase activity (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 performed with 32P-labeled p53 consensus oligonucleotide and 4 µg of nuclear protein purified from sham-FIR control MCF-7 and MCF+FIR cells. Lanes 0, without 5 Gy of IR. p53/DNA binding activity was estimated by densitometry (representative of three experiments)

 
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 MCF+FIR cells. Total p53 in cytoplasm showed little difference except a slight increase in 5-Gy-treated MCF+FIR cells (Fig. 3A)Citation . In contrast, the basal and IR-induced p53 (Thr 55) was enhanced in both the cytoplasm and the nucleus of MCF+FIR 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)Citation , suggesting an enhanced p53 degradation in control MCF-7 cells. This inhibitory function appears to be down-regulated in IR-derived MCF+FIR cells.



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  		Fig. 3. p53 and phosphorylated p53 [P-p53 (Thr 55)] after a single dose of 5 Gy. A, Western blotting was performed with the indicated antibody of p53 (sc-4246) or phosphorylated p53 [P-p53 (Thr 55), sc-12904-R] and 20 µg of cytoplasmic or nuclear proteins isolated from sham-fractionated ionizing radiation (FIR) control MCF-7 and MCF+FIR cells with or without 5 Gy of irradiation. B, relative levels of cytoplasmic and nuclear p53 proteins estimated by densitometric analysis (representative of three experiments).

 
p53-Responsive Genes Detected by DNA Microarray Analysis.
DNA microarray analysis was then applied to detect effector genes differently regulated in MCF+FIR versus sham-FIR control cells. We have reported a cluster of effector genes activated in the radioresistance induced by expression of MnSOD and/or FIR (15) . The present study shows gene expression levels in MCF+FIR versus the sham-FIR control cells using 588-1176 grouped human gene fragments (Clontech Atlas Human Cancer cDNA). Each gene was pair-wise analyzed by comparison of the expression level from MCF+FIR to the level of sham-FIR control MCF-7 cells. Genes found to be down-regulated (<2-fold decrease compared with MCF-7; Table 2Citation ) or up-regulated (>2-fold increase compared with MCF-7) were grouped by their functions and were ranked with gene expression levels (Table 3)Citation . A group of signaling elements associated with p53 regulation and signaling apoptosis and/or antiapoptosis including MDM2 (3.8-fold), p21 (4.6-fold), INKA-ARF (3.9-fold), IAP3 (4.1-fold), Cyclin B1 (3.3-fold), Cyclin D1 (3.4-fold), and others were up-regulated in MCF+FIR cells (marked in the list of Table 3Citation ). p14ARF, an alternative gene product of INKARF (39) has been indicated to act as a negative control of MDM2 and thus controls MDM2-mediated p53 regulation.


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  		Table 2 Down-regulated genes in comparison of MCF + fractionated ionizing radiation (FIR) versus sham-FIR control MCF-7 cellsa

 

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  		Table 3 Up-regulated genes in comparison of MCF + fractionated ionizing radiation (FIR) versus sham-FIR control MCF-7 cellsa

 
Because p21, MDM2, and p14ARF are closely linked with p53 regulation, we further analyzed the time course changes of the protein levels of p21, MDM2, and p14ARF and their nuclear distribution after IR.

p21 Induced by IR in Both Sham-FIR Control and MCF+FIR Cells.
To confirm the protein levels of p21 expression detected by microarray analysis, Western blot was performed using cytoplasmic and nuclear proteins of sham-FIR control and MCF+FIR cells with or without 5 Gy of IR (Fig. 4)Citation . Although the basal p21 protein expression was slightly increased (in agreement with up-regulated gene transcripts shown in Table 3Citation ) in the cytoplasm and nucleus of MCF+FIR cells (2-fold in cytoplasmic and 3-fold in nuclear proteins), no difference was detected in p21 induction and nuclear translocation after 5 Gy of IR (Fig. 4)Citation .



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  		Fig. 4. No difference in ionizing radiation (IR)-induced p21 protein expression and nuclear distribution. A, Western blotting was performed with 20 µg of cytoplasmic or nuclear proteins isolated from sham-fractionated IR (FIR) control MCF-7 and MCF+FIR cells at times indicated after 5 Gy of irradiation. The nonspecific bands (NS) were included to show equal loading of proteins (representative of three experiments). B, relative levels of cytoplasmic and nuclear p21 proteins estimated by the ratio of p21:NS bands measured with densitometry.

 
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 MCF+FIR cells (3.8-fold; Table 3Citation ). To get into the inside mechanisms underlying p53 activation in MCF+FIR 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 MCF+FIR cells but not in the sham-FIR control cells (Fig. 5Citation A, 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. 5Citation A, Lanes 5–8). In contrast, the cytoplasmic MDM2 was markedly enhanced (~7-fold at 3 h) in sham-FIR control cells (Fig. 5Citation A, 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 MCF+FIR cells with or without IR (Fig. 5B)Citation .



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  		Fig. 5. MDM2 expression and nuclear translocation were activated in sham-fractionated ionizing radiation (FIR) control MCF-7 but not in MCF+FIR cells. A, cytoplasmic MDM2 was induced by IR in sham-FIR control MCF-7 but not in MCF+FIR cells. Western blot was performed with 20 µg of protein from control MCF-7 and MCF+FIR cells at the indicated time intervals after 5 Gy of irradiation. MDM2 was visualized with MDM2 antibody, and expression levels were estimated by the ratio of MDM2:nonspecific bands (NS) measured with densitometry (lower panel, representative of three experiments). B, MDM2 was detected in the nucleus of sham-FIR control MCF-7 but not in MCF+FIR cells. Nuclear MDM2 was measured by Western blot with 20 µg of nuclear proteins purified from sham-FIR control MCF-7 or MCF+FIR cells 24 h after treatment with (+) or without (-) 5 Gy of IR. The nuclear proteins were separated on 12.5% acrylamide gel, and MDM2 was detected by MDM2 antibody. Nonspecific bands (NSs) were indicated as loading controls (representative of three experiments). C, MDM2 was detected in the p53/DNA complex of sham-FIR control MCF-7 but absent in the p53/DNA complex of MCF+FIR cells. Supershifting of MDM2 was performed with 32P-labeled p53 consensus oligonucleotide and 4 µg of nuclear proteins purified from sham-FIR control MCF-7 and MCF+FIR cells 24 h with (+) or without (-) 5 Gy of IR. The protein/DNA complex was then separated by gel electrophoresis. Arrows, p53/DNA complex and the shifted MDM2 proteins. Lower panel, the estimated ratio of MDM2 super-shifted bands:the level of p53/DNA complex (representative of three experiments).

 
We then asked the question of whether the nuclear-distributed MDM2 combines to p53/DNA complexes leading to a feed-back control of p53 activation. Supporting this concept, supershifting analysis with antibody to MDM2 demonstrated that MDM2 was present in the p53/DNA complex of the sham-FIR control cells 24 h after 5 Gy of IR (Fig. 5Citation C, Lanes 1 and 2), and MDM2 was not detected in the p53/DNA complex of MCF+FIR cells with or without 5 Gy of IR (Fig. 5Citation C, Lanes 3 and 4). These results demonstrate that MDM2/p53 binding occurs in the nucleus of control MCF-7 cells, indicating a feed-back control of p53 by MDM2 in IR-induced stress response. This regulation mechanism appears to be down-regulated in chronic IR-derived MCF+FIR cells. Lack of phosphorylated p53 (Thr 55) in control cells, but increased in MCF+FIR cells (Fig. 3)Citation , laid a support for this hypothesis.

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 MCF+FIR cells (Table 3)Citation .

The increased p14ARF that reduces p53 degradation by MDM2, may contribute to the high p53 level of protein in MCF+FIR cells. p14ARF mRNA and protein levels were analyzed with or without 5 Gy of IR (Fig. 6)Citation . Using published primers of p14ARF (43) , reverse transcription-PCR showed that basal p14ARF transcripts were very low but were enhanced by 5 Gy of IR (3-fold) in sham-FIR control cells (Fig. 6Citation A, Lanes 1 and 2). However, basal p14ARF transcripts were much more highly increased (~9-fold) and were maintained at a similar level in MCF+FIR cells after 5 Gy of IR (Fig. 6Citation A, Lanes 3 and 4). Consistent with the increased transcripts, p14ARF proteins that were detected in cytoplasmic and nuclear extracts were 6–8-fold higher in MCF+FIR cells compared with the levels of sham-FIR control cells (Fig. 6, B and C)Citation . These results suggest that the expression of p14ARF and the inhibition of MDM2 work together to maintain p53 protein in the nucleus causing a high responsiveness of p53 in chronic IR-derived radioresistant cells.



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  		Fig. 6. p14ARF mRNA and protein levels were increased in MCF+fractionated ionizing radiation (FIR) cells. A, p14ARF mRNA level was increased in MCF+FIR cells. p14ARF transcripts were detected by reverse transcription-PCR using the published p14ARF primers shown in "Materials and Methods" and 5 µg of total RNA of sham-FIR control (C) MCF-7 and MCF+FIR cells. Gene fragments were enhanced for 25 cycles, and relative levels of transcripts were estimated by densitometric analysis and were presented as arbitrary units after normalization to the level of GAPDH (representative of three experiments). B, Western blot was performed with p14ARF antibody and 20 µg of cytoplasmic or nuclear proteins of control MCF-7 and MCF+FIR cells 24 h after 5 Gy of IR (representative of three experiments). C, the cytoplasmic and nuclear p14ARF levels were estimated by the ratio of p14ARF:Actin bands measured with densitometry.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present findings demonstrate that p53 is actively involved in human breast carcinoma MCF+FIR cells that showed a transient radioresistance after chronic exposure to IR. p53 transcriptional activity was increased with little recruitment of p53 in DNA binding; and p53-modulating protein MDM2 was inhibited and MDM2-inhibitory protein p14ARF was activated in MCF+FIR cells. These results suggest that p53-regulating elements MDM2 and p14ARF play a key role in the up-regulation of the p53 activity under chronic stresses with IR.

p53 signaling network has been shown to play a central role in maintaining the genomic integrity and protecting cells against IR-induced damage (18 , 26 , 27) . The present results indicate an elevated "constitutive" DNA binding and transcriptional activity of p53 in IR-derived MCF+FIR cells that showed an enhanced resistance to IR-induced apoptosis. It has been well accepted that activation of p53 is able to adjust the cell cycle time to allow cells to repair the damaged DNA induced by IR (44) . IR-induced cell cycle delay is attributed to the activation of p53 (45) , which induces cyclin-dependent kinase-inhibitory protein p21, which is, in turn, required for specific cell cycle checkpoints (30 , 46) . However, p21 showed little response to anticancer agent-induced stress in some tumor cells. Loss of p21 results in an induction of apoptosis, but no difference was found in the clonogenic survival (47 , 48) , in IR-induced S-phase checkpoint (41) , or in DNA repair responses to UV radiation (21) . We report here that, although p53 and p21 were activated in IR-derived MCF+FIR cells, no difference was detected in p21 protein and nuclear translocation in control and MCF+FIR cells after IR. In addition, our published results have shown that blocking p21 by antisense transfection did not enhance the radiosensitivity of IR-derived MCF+FIR cells (9) . These results argue that although p21 transcription can be up-regulated because of p53 activation, p21 protein may not function as a rate-limiting element in chronic IR-induced stress response.

The reported (49) and present data (Table 3Citation ; Fig. 5Citation ) demonstrate that MDM2 transcripts are increased by IR. To determine MDM2 response in IR-derived cell, however, we found that MDM2 was inhibited in MCF+FIR cells after the stress with a single dose of IR. In contrast, MDM2 was induced (~7-fold) by IR (Fig. 5A)Citation and MDM2 was detected in the p53/DNA binding complex of sham-FIR control but not IR-derived MCF+FIR cells (Fig. 5, B and C)Citation . 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. Further evidence is provided by the fact that MDM2 was directly detected in p53/DNA complex in control MCF-7 but not in IR-derived MCF+FIR cells (Figs. 5, B and C)Citation .

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.

An important finding in the present microarray and protein expression studies is the activation of p14ARF. In mammalian cells, the INK4{alpha}-p14ARF locus has been identified to encode two different cell cycle inhibitors (p16INK4{alpha} and p14ARF) by alternative splicing (51 , 52) . It has been suggested that ARF family proteins regulate p53 activity (36 , 53) via a negative regulation by MDM2 (53 , 54) . p14ARF is also required for MDM2 degradation by small ubiquitin-like protein (SUMO; Ref. 42 ). In the present study, we found that p14ARF was activated in MCF+FIR cells that showed down-regulation of MDM2 and up-regulation of p14ARF. As a result, p53 luciferase transcription was much higher in MCF+FIR cells than control cells after IR. Correspondingly, expression of p14ARF and MDM2 was found to be oppositely regulated in sham-FIR control cells after IR. This difference between control and chronic IR-derived cells suggests an adjustment of p14ARF and MDM2 proteins, which appears to be required for maintaining a relatively high level of p53 responsiveness in IR-derived cells. This adjustment may contribute to a quick transactivation of p53 without the recruitment of p53 into the nucleus.

Inhibition of p53 has radiosensitized human diploid fibroblasts (55) and expression of p53 is associated with local failure of neck and head tumors in radiotherapy (56) . Although a direct linking between p53 activation and radioresistance has not been identified, transcription factor NF-{kappa}B, evidently related to radioresistance (10 , 57 , 58) , is associated with p53 activation. The inhibition of NF-{kappa}B abrogates p53-induced apoptosis (59) , and expression of p65, the key subunit of NF-{kappa}B, activates p53 gene promoter activity (60) . We have reported that both p53 and NF-{kappa}B are activated by IR in MCF-7 cells (9) , and NF-{kappa}B activation is causally related to IR-derived radioresistance (10 , 15) . The coordinated modulation of p53 and NF-{kappa}B pathways has been indicated in IR-induced response in human malignant melanoma cells (61) .

When we follow this line of reasoning, p53 appears to participate the radioresistance-signaling network leading to the activation of NF-{kappa}B. In addition, p53 and NF-{kappa}B likely regulate the function of Ku86 a subunit of DNA-dependent protein kinase (DNA-PK) and a key protein in DNA repair (62) . Our microarray data showed that, compared with sham-FIR control cells, Ku86 transcripts were up-regulated in IR-derived MCF+FIR cells (Table 3)Citation . Because Ku86 and its isoform KARP-1 can be regulated by p53 (63) and, importantly, DNA-dependent protein kinase is shown to phosphorylate I{kappa}B{alpha} for NF-{kappa}B activation (64) , NF-{kappa}B can be activated in IR-derived cells by Ku86 as a result of p53 activation. Such a Ku86-mediated connection between p53 and NF-{kappa}B networks may function as a potential molecular adjustment in IR-derived radioresistance.

In conclusion, MCF-7 cells exposed to chronic IR showed increased basal and IR-induced p53 activity. MDM2 was down-regulated and p14ARF, an inhibitor of MDM2, was up-regulated in IR-derived MCF+FIR cells, and these two proteins were oppositely regulated in the sham-FIR control cells. These results suggest that adjustment in p14ARF and MDM2 expression is required to maintain a high responsiveness of p53 in chronic IR-derived radioresistant cells.


   ACKNOWLEDGMENTS
 
We thank Zhi-Min Yuan at Harvard University for insightful discussion, Zhongkui Li at University of Texas M. D. Anderson Medical Center for assistance in DNA microarray analysis, Jeffrey Longmate at City of Hope National Medical Center for statistical analysis, and Kurt Fried for reading the manuscript.


   FOOTNOTES
 
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.

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: (626) 301-8892; E-mail: jjli{at}coh.org

Received 4/10/03. Revised 9/19/03. Accepted 10/20/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hill B. T., Shellard S. A., Hosking L. K., Fichtinger-Schepman A. M., Bedford P. Enhanced DNA repair and tolerance of DNA damage associated with resistance to cis-diamminedichloroplatinum (II) after in vitro exposure of a human teratoma cell line to fractionated X-irradiation. Int. J. Radiat. Oncol. Biol. Phys., 19: 75-83, 1990.[Medline]
  2. Joiner M. C., Lambin P., Malaise E. P., Robson T., Arrand J. E., Skov K. A., Marples B. Hypersensitivity to very-low single radiation doses: its relationship to the adaptive response and induced radioresistance. Mutat. Res., 358: 171-183, 1996.[CrossRef][Medline]
  3. Eichholtz-Wirth H., Marx K. Clonal variability of radiation-induced cisplatin resistant HeLa cells. Anticancer Res., 18: 2989-2991, 1998.[Medline]
  4. Azzam E. I., de Toledo S. M., Raaphorst G. P., Mitchel R. E. Low-dose ionizing radiation decreases the frequency of neoplastic transformation to a level below the spontaneous rate in C3H 10T1/2 cells. Radiat. Res., 146: 369-373, 1996.[Medline]
  5. Boothman D. A., Odegaard E., Yang C. R., Hosley K., Mendonca M. S. Molecular analyses of adaptive survival responses (ASRs): role of ASRs in radiotherapy. Hum. Exp. Toxicol., 17: 448-453, 1998.[Abstract/Free Full Text]
  6. Feinendegen L. E., Bond V. P., Sondhaus C. A., Altman K. I. Cellular signal adaptation with damage control at low doses versus the predominance of DNA damage at high doses. C. R. Acad. Sci. Ser. III, 322: 245-251, 1999.[Medline]
  7. Feinendegen L. E. Reactive oxygen species in cell responses to toxic agents. Hum. Exp. Toxicol., 21: 85-90, 2002.[Abstract/Free Full Text]
  8. Russell J., Wheldon T. E., Stanton P. A radioresistant variant derived from a human neuroblastoma cell line is less prone to radiation-induced apoptosis. Cancer Res., 55: 4915-4921, 1995.[Abstract/Free Full Text]
  9. Li Z., Xia L., Lee M. L., Khaletskiy A., Wang J., Wong J. Y. C., Li J. J. Effector genes altered in MCF-7 human breast cancer cells after exposure to fractionated ionizing radiation. Radiat. Res., 155: 543-553, 2001.[Medline]
  10. Chen X., Shen B., Xia L., Khaletzkiy A., Chu D., Wong J. Y., Li J. J. Activation of nuclear factor {kappa}B in radioresistance of TP53-inactive human keratinocytes. Cancer Res., 62: 1213-1221, 2002.[Abstract/Free Full Text]
  11. Fornace A. J., Jr., Amundson S. A., Bittner M., Myers T. G., Meltzer P., Weinsten J. N., Trent J. The complexity of radiation stress responses: analysis by informatics and functional genomics approaches. Gene. Expr., 7: 387-400, 1999.[Medline]
  12. Schmidt-Ullrich R. K., Contessa J. N., Dent P., Mikkelsen R. B., Valerie K., Reardon D. B., Bowers G., Lin P. S. Molecular mechanisms of radiation-induced accelerated repopulation. Radiat. Oncol. Investig., 7: 321-330, 1999.[CrossRef][Medline]
  13. McBride W. H., Pajonk F., Chiang C. S., Sun J. R. NF-{kappa}B, cytokines, proteasomes, and low-dose radiation exposure. Mil. Med., 167: 66-67, 2002.[Medline]
  14. Li Z., Khaletskiy A., Wang J., Wong J. Y., Oberley L. W., Li J. J. Genes regulated in human breast cancer cells overexpressing manganese-containing superoxide dismutase. Free Radic. Biol. Med., 30: 260-267, 2001.[CrossRef][Medline]
  15. Guo G., Yan-Sanders Y., Lyn-Cook B. D., Wang T., Tamae D., Ogi J., Khaletskiy A., Li Z., Weydert C., Longmate J. A., Huang T. T., Spitz D. R., Oberley L. W., Li J. J. Manganese superoxide dismutase mediated gene expression in radiation-induced adaptive responses. Mol. Cell. Biol., 23: 2362-2378, 2003.[Abstract/Free Full Text]
  16. Kastan M. B., Onyekwere O., Sidransky D., Vogelstein B., Craig R. W. Participation of p53 protein in the cellular response to DNA damage. Cancer Res., 51: 6304-6311, 1991.[Medline]
  17. Giaccia A. J., Kastan M. B. The complexity of p53 modulation: emerging patterns from divergent signals. Genes Dev., 12: 2973-2983, 1998.[Free Full Text]
  18. Kastan M. B., Zhan Q., El-Deiry W. S., Carrier F., Jacks T., Walsh W. V., Plunkett B. S., Vogelstein B., Fornace A. J., Jr. A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell, 71: 587-597, 1992.[CrossRef][Medline]
  19. Kuerbitz S. J., Plunkett B. S., Walsh W. V., Kastan M. B. Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Proc. Natl. Acad. Sci. USA, 89: 7491-7495, 1992.[Abstract/Free Full Text]
  20. Lowe S. W., Schmitt E. M., Smith S. W., Osborne B. A., Jacks T. p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature (Lond.), 362: 847-849, 1993.[CrossRef][Medline]
  21. Smith M. L., Ford J. M., Hollander M. C., Bortnick R. A., Amundson S. A., Seo Y. R., Deng C. X., Hanawalt P. C., Fornace A. J., Jr. p53-mediated DNA repair responses to UV radiation: studies of mouse cells lacking p53, p21, and/or gadd45 genes. Mol. Cell. Biol., 20: 3705-3714, 2000.[Abstract/Free Full Text]
  22. Tokino T., Thiagalingam S., el-Deiry W. S., Waldman T., Kinzler K. W., Vogelstein B. p53 tagged sites from human genomic DNA. Hum. Mol. Genet., 3: 1537-1542, 1994.[Abstract/Free Full Text]
  23. Amundson S. A., Bittner M., Chen Y., Trent J., Meltzer P., Fornace A. J., Jr. Fluorescent cDNA microarray hybridization reveals complexity and heterogeneity of cellular genotoxic stress responses. Oncogene, 18: 3666-3672, 1999.[CrossRef][Medline]
  24. Bouvard V., Zaitchouk T., Vacher M., Duthu A., Canivet M., Choisy-Rossi C., Nieruchalski M., May E. Tissue and cell-specific expression of the p53-target genes: bax, fas, mdm2 and waf1/p21, before and following ionising irradiation in mice. Oncogene, 19: 649-660, 2000.[CrossRef][Medline]
  25. Haas-Kogan D. A., Kogan S. S., Yount G., Hsu J., Haas M., Deen D. F., Israel M. A. p53 function influences the effect of fractionated radiotherapy on glioblastoma tumors. Int. J. Radiat. Oncol. Biol. Phys., 43: 399-403, 1999.[CrossRef][Medline]
  26. Sharpless N. E., DePinho R. A. p53. Good Cop/Bad Cop. Cell, 110: 9-12, 2002.
  27. Nelson W. G., Kastan M. B. DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways. Mol. Cell. Biol., 14: 1815-1823, 1994.[Abstract/Free Full Text]
  28. Haupt Y., Maya R., Kazaz A., Oren M. Mdm2 promotes the rapid degradation of p53. Nature (Lond.), 387: 296-299, 1997.[CrossRef][Medline]
  29. Kubbutat M. H., Jones S. N., Vousden K. H. Regulation of p53 stability by Mdm2. Nature (Lond.), 387: 299-303, 1997.[CrossRef][Medline]
  30. Bunz F., Dutriaux A., Lengauer C., Waldman T., Zhou S., Brown J. P., Sedivy J. M., Kinzler K. W., Vogelstein B. Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science (Wash. DC), 282: 1497-1501, 1998.[Abstract/Free Full Text]
  31. Ashcroft M., Taya Y., Vousden K. H. Stress signals utilize multiple pathways to stabilize p53. Mol. Cell. Biol., 20: 3224-3233, 2000.[Abstract/Free Full Text]
  32. Lu W., Pochampally R., Chen L., Traidej M., Wang Y., Chen J. Nuclear exclusion of p53 in a subset of tumors requires MDM2 function. Oncogene, 19: 232-240, 2000.[CrossRef][Medline]
  33. Gu J., Nie L., Wiederschain D., Yuan Z. M. Identification of p53 sequence elements that are required for MDM2-mediated nuclear export. Mol. Cell. Biol., 21: 8533-8546, 2001.[Abstract/Free Full Text]
  34. Lohrum M. A., Woods D. B., Ludwig R. L., Balint E., Vousden K. H. C-terminal ubiquitination of p53 contributes to nuclear export. Mol. Cell. Biol., 21: 8521-8532, 2001.[Abstract/Free Full Text]
  35. Tao W., Levine A. J. P19(ARF) stabilizes p53 by blocking nucleo-cytoplasmic shuttling of Mdm2. Proc Natl. Acad. Sci. USA, 96: 6937-6941, 1999.[Abstract/Free Full Text]
  36. Zhang Y., Xiong Y. A p53 amino-terminal nuclear export signal inhibited by DNA damage-induced phosphorylation. Science (Wash. DC), 292: 1910-1915, 2001.[Abstract/Free Full Text]
  37. Honda R., Yasuda H. Association of p19(ARF) with Mdm2 inhibits ubiquitin ligase activity of Mdm2 for tumor suppressor p53. EMBO J., 18: 22-27, 1999.[CrossRef][Medline]
  38. Midgley C. A., Desterro J. M., Saville M. K., Howard S., Sparks A., Hay R. T., Lane D. P. An N-terminal p14ARF peptide blocks Mdm2-dependent ubiquitination in vitro and can activate p53 in vivo. Oncogene, 19: 2312-2323, 2000.[CrossRef][Medline]
  39. DiDonato J. A., Hayakawa M., Rothwarf D. M., Zandi E., Karin M. A cytokine-responsive I{kappa}B kinase that activates the transcription factor NF-{kappa}B. Nature (Lond.), 388: 548-554, 1997.[CrossRef][Medline]
  40. Sun Y., Dong Z., Nakamura K., Colburn N. H. Dosage-dependent dominance over wild-type p53 of a mutant p53 isolated from nasopharyngeal carcinoma. FASEB J., 7: 944-950, 1993.[Abstract]
  41. Yu C. R., Wang L., Khaletskiy A., Farrar W. L., Larner A., Colburn N. H., Li J. J. Activation of STAT3 in IL-6 induced transformation response in mouse skin epithelial cells. Oncogene, 21: 3940-3960, 2002.
  42. Xirodimas D. P., Chisholm J., Desterro J. M., Lane D. P., Hay R. T. P14ARF promotes accumulation of SUMO-1 conjugated (H)Mdm2. FEBS Lett., 528: 207-211, 2002.[CrossRef][Medline]
  43. Eymin B., Gazzeri S., Brambilla C., Brambilla E. Mdm2 overexpression and p14(ARF) inactivation are two mutually exclusive events in primary human lung tumors. Oncogene, 21: 2750-2761, 2002.[CrossRef][Medline]
  44. Di Leonardo A., Linke S. P., Clarkin K., Wahl G. M. DNA damage triggers a prolonged p53-dependent G1 arrest and long-term induction of Cip1 in normal human fibroblasts. Genes Dev., 8: 2540-2551, 1994.[Abstract/Free Full Text]
  45. Ko L. J., Prives C. p53: puzzle and paradigm. Genes Dev., 10: 1054-1072, 1996.[Free Full Text]
  46. El-Deiry W. S. Regulation of p53 downstream genes. Semin. Cancer Biol., 8: 345-357, 1998.[CrossRef][Medline]
  47. Waldman T., Lengauer C., Kinzler K. W., Vogelstein B. Uncoupling of S phase and mitosis induced by anticancer agents in cells lacking p21. Nature (Lond.), 381: 713-716, 1996.[CrossRef][Medline]
  48. Wouters B. G., Giaccia A. J., Denko N. C., Brown J. M. Loss of p21Waf1/Cip1 sensitizes tumors to radiation by an apoptosis- independent mechanism. Cancer Res., 57: 4703-4706, 1997.[Abstract/Free Full Text]
  49. Amundson S. A., Do K. T., Fornace A. J., Jr. Induction of stress genes by low doses of {gamma} rays. Radiat. Res., 152: 225-231, 1999.[Medline]
  50. Gu J., Chen D., Rosenblum J., Rubin R. M., Yuan Z. M. Identification of a sequence element from p53 that signals for Mdm2-targeted degradation. Mol. Cell. Biol., 20: 1243-1253, 2000.[Abstract/Free Full Text]
  51. Serrano M., Hannon G. J., Beach D. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature (Lond.), 366: 704-707, 1993.[CrossRef][Medline]
  52. Quelle D. E., Zindy F., Ashmun R. A., Sherr C. J. Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest. Cell, 83: 993-1000, 1995.[CrossRef][Medline]
  53. Zhang Y., Xiong Y., Yarbrough W. G. ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways. Cell, 92: 725-734, 1998.[CrossRef][Medline]
  54. Vousden K. H. p53: death star. Cell, 103: 691-694, 2000.[CrossRef][Medline]
  55. Tsang N. M., Nagasawa H., Li C., Little J. B. Abrogation of p53 function by transfection of HPV16 E6 gene enhances the resistance of human diploid fibroblasts to ionizing radiation. Oncogene, 10: 2403-2408, 1995.[Medline]
  56. Couture C., Raybaud-Diogene H., Tetu B., Bairati I., Murry D., Allard J., Fortin A. p53 and Ki-67 as markers of radioresistance in head and neck carcinoma. Cancer (Phila.), 94: 713-722, 2002.
  57. Bradbury C. M., Markovina S., Wei S. J., Rene L. M., Zoberi I., Horikoshi N., Gius D. Indomethacin-induced radiosensitization and inhibition of ionizing radiation-induced NF-{kappa}B activation in HeLa cells occur via a mechanism involving p38 MAP kinase. Cancer Res., 61: 7689-7696, 2001.[Abstract/Free Full Text]
  58. Wang T., Zhang X., Li J. J. The role of NF-{kappa}B in the regulation of cell stress responses. Int. Immunopharmacol., 2: 1509-1520, 2002.[CrossRef][Medline]
  59. Ryan K. M., Ernst M. K., Rice N. R., Vousden K. H. Role of NF-{kappa}B in p53-mediated programmed cell death. Nature (Lond.), 404: 892-897, 2000.[CrossRef][Medline]
  60. Wu H., Lozano G. NF-{kappa}B activation of p53. A potential mechanism for suppressing cell growth in response to stress. J. Biol. Chem., 269: 20067-20074, 1994.[Abstract/Free Full Text]
  61. Yang C. R., Wilson-Van Patten C., Planchon S. M., Wuerzberger-Davis S. M., Davis T. W., Cuthill S., Miyamoto S., Boothman D. A. Coordinate modulation of Sp1, NF-{kappa}B, and p53 in confluent human malignant melanoma cells after ionizing radiation. FASEB J., 14: 379-390, 2000.[Abstract/Free Full Text]
  62. Li G., Nelsen C., Hendrickson E. A. Ku86 is essential in human somatic cells. Proc. Natl. Acad. Sci. USA, 99: 832-837, 2002.[Abstract/Free Full Text]
  63. Braastad C. D., Leguia M., Hendrickson E. A. Ku86 autoantigen related protein-1 transcription initiates from a CpG island and is induced by p53 through a nearby p53 response element. Nucleic Acids Res., 30: 1713-1724, 2002.[Abstract/Free Full Text]
  64. Liu L., Kwak Y. T., Bex F., Garcia-Martinez L. F., Li X. H., Meek K., Lane W. S., Gaynor R. B. DNA-dependent protein kinase phosphorylation of I{kappa}B {alpha} and I{kappa}B ß regulates NF-{kappa}B DNA binding properties. Mol. Cell. Biol., 18: 4221-4234, 1998.[Abstract/Free Full Text]



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