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Clinical Cancer Research 13, 4245, July 15, 2007. doi: 10.1158/1078-0432.CCR-06-2890
© 2007 American Association for Cancer Research
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Cancer Therapy: Preclinical

Regulation of p53 Expression in Response to 5-Fluorouracil in Human Cancer RKO Cells

Jingfang Ju1, John C. Schmitz2,3, Bo Song1, Kenji Kudo1 and Edward Chu2,3

Authors' Affiliations: 1 Cancer Genomics Laboratory, Mitchell Cancer Institute-USA, Mobile, Alabama and 2 Department of Medicine and Pharmacology, Yale Cancer Center, Yale University School of Medicine and 3 VA Connecticut Healthcare System, New Haven, Connecticut

Requests for reprints: Jingfang Ju, Mitchell Cancer Institute-USA, MSB2316, 307 N. University Boulevard, Mobile, AL 36688. Phone: 251-460-7393; Fax: 251-460-6994; E-mail: jju{at}usouthal.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: The purpose of the study is to investigate the regulation of p53 expression in response to 5-fluorouracil (5-FU) in human colon cancer cells.

Experimental Design: Human colon cancer RKO cells were used as our model system. The levels of p53 expression and p53 protein stability in response to 5-FU and doxorubicin were investigated. In addition, the acetylation and phosphorylation status of p53 after 5-FU and doxorubicin treatment was analyzed by Western immunoblot analysis.

Results: Treatment of human colon cancer RKO cells with 10 µmol/L 5-FU resulted in significantly increased levels of p53 protein with maximal induction observed at 24 h. The level of acetylated p53 after 5-FU exposure remained unchanged, whereas the phosphorylated form of p53 was expressed only after 24 h drug treatment. Northern blot analysis revealed no change in p53 mRNA levels after 5-FU treatment. No differences were observed in the half-life of p53 protein in control and 5-FU–treated cells, suggesting that the increase in p53 was the direct result of newly synthesized protein. In contrast, the maximal induction of p53, in response to doxorubicin, occurred at an earlier time point (4 h) when compared with cells treated with 5-FU (24 h). No corresponding change in p53 mRNA was observed. Levels of both the acetylated and phosphorylated forms of p53 were markedly increased upon doxorubicin exposure when compared with treatment with 5-FU, resulting in a significantly prolonged half-life of p53 (120 versus 20 min).

Conclusion: These results, taken together, suggest that the regulatory mechanisms controlling p53 expression, in response to a cellular stress, are complex and are dependent upon the specific genotoxic agent. With regard to 5-FU, we show that translational regulation is an important process for controlling p53 expression. Studies are under way to define the specific mechanism(s) that control 5-FU–mediated translational regulation of p53.

p53 is a well-established tumor suppressor protein, and mutations in the p53 gene are among the most common genetic alterations observed in human tumor samples. It is estimated that >50% of all human cancers express either a mutant or altered form of p53 (1–3). In addition to preventing tumor growth, p53 plays a key role in cell cycle regulation and in the cellular response to cytotoxic stress and DNA damage (4–6). Wild-type p53 is induced in response to a host of genotoxic and environmental stresses, including {gamma} and UV irradiation, chemotherapeutic agents, hypoxia, heat, or alterations in intracellular nucleotide pools (4–7). Once p53 is induced, a host of target genes are then transcriptionally activated, including p21, GADD45, bax, and bcl-2. Induction of p21, in turn, leads to cell cycle arrest at both G1 and G2 checkpoints. This function is felt to be essential in preserving the integrity of the cellular genome in response to treatment with cytotoxic agents. In addition to mediating cell cycle arrest, p53 is a potent inducer of the process of apoptosis and the program of cell death (8–10). At this time, the specific signals required for p53-mediated cell cycle arrest and/or apoptosis remain complex. However, the final pathway selected seems to depend upon a host of physiologic conditions, the cellular context and environment and the specific cytotoxic and/or cellular stress.

The mechanisms governing the expression of p53, in response to genotoxic stress, are complex. Much of the initial work suggested that the induction of p53 was controlled at the posttranscriptional level, primarily through increased stability of the protein (11). Posttranslational modifications at both the amino and carboxy termini of p53 are central to activating its DNA-specific binding activity (12). In response to DNA damage, p53 is phosphorylated at serine 15, which then mediates activation of p53 transcriptional function (13, 14). More recently, it has been shown that UV irradiation results in phosphorylation at serine 389 in the COOH terminus. In addition to phosphorylation, acetylation at the COOH terminus is required for p53 function (15–17). Recent work has also shown interactive cross-talk between the NH2 and COOH termini of p53, as phosphorylation at the amino terminus helps to direct acetylation in the carboxy terminus (18). These posttranslational events inhibit the interaction between p53 and mdm-2 interaction, and in so doing, prevent the subsequent degradation of p53 by the ubiquitin pathway.

Several other regulatory processes, including transcriptional and/or translational events, have been identified as mediating the induced expression of p53 (19, 20). Recent work suggests that p53 may regulate its own mRNA translation by binding to the 3' untranslated region of the mRNA, resulting in translational repression (21–23). In addition to the process of p53 translational autoregulation, results from our own laboratory showed that the folate-dependent enzyme thymidylate synthase (TS), in its capacity as an RNA binding protein, can regulate the expression of p53 by directly binding to a sequence within the protein-coding region of p53 mRNA (24, 25). Both in vitro and in vivo model systems have now confirmed that this RNA-protein interaction leads to translation repression of p53 mRNA, with subsequent downstream effects on cell cycle control and apoptosis in response to exposure to a host of anticancer agents (24, 25).

In this report, we show that the expression of p53 in human colon cancer RKO cells, in response to the fluoropyrimidine 5-fluorouracil (5-FU), is regulated primarily at the translational level. In contrast, the expression of p53 after treatment with doxorubicin, a cytotoxic agent with a different molecular target than 5-FU, is controlled by posttranslational modification of the protein, including acetylation and phosphorylation. These studies suggest that the regulatory mechanisms involved in controlling p53 expression in response to a cytotoxic stress are complex and may depend, in large measure, upon the specific cytotoxic agent under investigation.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Chemicals. 5-FU and doxorubicin were purchased from Sigma Chemical Co. [35S]methionine (specific activity, 1 mCi/mL) was purchased from NEN Life Science. All other chemicals were of the highest grade possible and were obtained from Sigma Chemical Co.

Cell culture. The characteristics of the human colon cancer RKO cell line have been previously described (24). Cells were grown in RPMI 1640 containing 10% dialyzed fetal bovine serum (Life Technologies-Bethesda Research Laboratories).

Western immunoblot analysis. Cellular extracts were prepared as described previously (24, 25). Protein concentrations were determined by a protein assay (Bio-Rad Laboratories), and equivalent amounts of total cellular protein from each cell line were resolved by 12.5% SDS-PAGE, according to the method of Laemmli (26). The gels were then electroblotted onto nitrocellulose membranes. Primary antibody staining was done with the use of a mouse anti-p53 monoclonal antibody (Ab-2, Oncogene Science; 1:150 dilution), mouse anti–ß-actin monoclonal antibody (Amersham; 1:4,000 dilution), rabbit anti-phosphorylated p53 polyclonal antibody (1:1,000 dilution; New England Biolab), rabbit anti-acetylated lysine polyclonal antibody (New England Biolab). After primary antibody staining, filter membranes were incubated with a horseradish peroxidase–conjugated secondary antibody (Bio-Rad). Proteins were then visualized with a chemiluminescence detection system using the Super Signal substrate (Pierce), and protein bands were visualized by autoradiography. Quantitation of signal intensities was done by densitometry with a ScanJet Plus scanner and analyzed with NIH Image 1.59 software.

Isolation of nuclear and cytoplasmic p53. RKO cells were treated with 5-FU for 24 h, and cells were then washed thrice with ice-cold PBS. Nuclear and cytoplasmic fractions were isolated based upon the NE-PER Pierce isolation protocol (Pierce), and Western immunoblot analysis was done exactly as described above.

Isolation of total RNA and Northern blot analysis. Human colon cancer RKO cells were washed thrice with ice-cold PBS and harvested from 75-cm2 tissue culture flasks with a rubber policeman. Total cellular RNA was extracted according to the method of Chomczynski and Sacchi (27), and the RNA concentration was determined by UV spectrophotometry. Ten micrograms of RNA from each sample were denatured, resolved on a 1% agarose-formaldehyde gel, and then transferred onto a Nytran filter membrane (Schleicher and Schuell). A biotinylated antisense p53 RNA sequence was synthesized by in vitro transcription using the pTRI-p53–human transcription template (Ambion) and T7 RNA polymerase (Ambion). A biotinylated 28S RNA probe was synthesized in vitro by using the human pTRI-28S transcription template (Ambion) and T7 RNA polymerase. The filter membranes were then incubated with the respective biotinylated antisense RNA probes in the hybridization reaction. After overnight incubation at 65°C, filters were washed and processed using the Brightstar Biodetect kit (Ambion).

Measurement of p53 biosynthesis by immunoprecipitation analysis. Cells were washed twice with methionine-free RPMI 1640 containing 10% dialyzed fetal bovine serum. Cells were treated in the absence or presence of drug for 22 h and then pulse-labeled with 150 µCi [35S]methionine (specific activity, 1 mCi/mL) for 2 h in methionine-free RPMI 1640 containing 10% dialyzed fetal bovine serum. Radiolabel was removed, and fresh methionine-containing medium was added to the cell cultures. At various time points (0, 10, 20, 30, and 60 min), cells were harvested and processed. Cells were pelleted by centrifugation at 2,000 rpm for 5 min. The supernatant was removed, and cells were lysed in 0.5 mL ice-cold lysis buffer. Immunoprecipitation of p53 protein was done with an anti-p53 monoclonal antibody (Ab-2, Oncogene Science) and protein-A agarose (Life Technologies-Bethesda Research Laboratories) according to previously described methods (25, 28). Each sample contained 20 x 106 cpm of trichloroacetic acid–insoluble radioactivity. Samples were analyzed by autoradiography after electrophoresis on a 10% SDS-polyacrylamide gel. The relative levels of p53 protein were then determined by densitometric scanning using a ScanJet Plus scanner and analyzed with NIH Image 1.59 software. Protein half-life was calculated based on the trendline equation generated from Excel software and x1/2(min) = –intercept/2/slop.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Effect of 5-FU on p53 expression. Previous studies have shown that the expression of p53 protein is induced in response to various genotoxic stresses, including UV and {gamma} irradiation and various anticancer drugs (5–7, 11, 19, 20). In this study, we investigated the effect of the fluoropyrimidine 5-FU on p53 expression. For these experiments, the well-established RKO cell line was selected as our model system, as it expresses native wild-type p53. As seen in Fig. 1 , levels of p53 protein were significantly induced as early as 4 h by 2.5-fold after treatment of RKO cells with 5-FU. For these time-dependent experiments, a 5-FU concentration of 10 µmol/L was selected. This concentration of drug represents the IC90 value for 5-FU, as determined by growth inhibition studies (data not shown). Similar levels of p53 induction were observed after exposure to the IC50 concentration of 5-FU (1 µmol/L), as well as to 25 µmol/L, a dose which completely inhibited cell growth. Maximal induction of p53 protein was observed at 24 h after drug exposure, with nearly a 12-fold increase in protein expression (Fig. 1A). In contrast, the levels of control housekeeping proteins, such as ß-actin or {alpha}-tubulin (data not shown), were unchanged.


Figure 1
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  		Fig. 1. A, effect of 5-FU on expression of p53 protein. RKO cells were incubated with 10 µmol/L 5-FU for varying periods of time (0-24 h). Whole-cell extracts were prepared, equal amounts of cellular protein were resolved, and Western immunoblot analyses were done as described in Materials and Methods. B, effect of 5-FU on expression of p53 mRNA. RKO cells were incubated with 10 µmol/L 5-FU for varying periods of time (0-24 h). Equal amounts (10 µg) of total cellular RNA were incubated with a biotinylated antisense p53 RNA probe and processed as described in Materials and Methods. The filter membranes were then stripped of the p53 probe and rehybridized with a human 28S antisense RNA probe to control for loading and integrity of mRNA. C, densitometric analysis of the effect of 5-FU on expression of p53 protein and p53 mRNA levels.

 
A Northern RNA blot analysis was done to determine whether the increase in p53 protein was associated with a corresponding change in p53 mRNA levels. As seen in Fig. 1B, the levels of p53 mRNA were unchanged after 5-FU treatment when compared with control, non–drug-treated RKO cells. To control for loading and integrity of RNA, the expression of 28S RNA in parent and drug-treated cells was determined, and we found that the levels of ribosomal 28S RNA remained unchanged with drug treatment.

The experimental data from the Western immunoblot and Northern RNA blot analyses were then pooled to form a composite graph, as presented in Fig. 1C. As seen, there was a marked discordance between the levels of p53 protein and p53 mRNA in response to treatment with 5-FU. These results suggest that the 5-FU–associated induction of p53 protein was controlled at the posttranscriptional mechanism.

The respective nuclear and cytoplasmic fractions from whole-cell extracts were then isolated to determine whether treatment with 5-FU might alter the intracellular localization of p53. As seen in Fig. 2A , p53 protein was localized primarily to the nucleus under normal growth conditions. Treatment with 5-FU resulted in dramatically increased expression of p53, all of which was expressed entirely within the nucleus. This finding suggests that treatment with 5-FU did not give rise to a redistribution of the protein within the cell. As an important control, we observed that the expression of {alpha}-tubulin, a protein which is strictly localized to within the cytoplasmic compartment, was completely unaltered with 5-FU treatment and remained confined to the cytoplasm. In addition, topoisomerase I, which is localized within the nuclear compartment, was unchanged after 5-FU treatment. Similar findings were also observed after doxorubicin treatment (Fig. 2B).


Figure 2
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  		Fig. 2. Effect of 5-FU and doxorubicin on the cytoplasmic and nuclear fractions of p53. RKO cells were treated with 10 µmol/L 5-FU (A) or 5 µmol/L doxorubicin (B) for 24 h, and the respective cytoplasmic and nuclear cellular fractions were isolated as described in Materials and Methods. Western immunoblot analysis was done using either an anti-p53 monoclonal antibody, an anti-{alpha}-tubulin monoclonal antibody, or an anti-topoisomerase I (Topo I) monoclonal antibody.

 
Previous studies have shown that posttranslational processing of p53 takes place after exposure to {gamma} and UV irradiation (12–18). These modifications of p53 protein include acetylation and phosphorylation, and both events are felt to effect directly on the transcriptional activity of p53 and its subsequent downstream biological effects. With this in mind, we investigated the level of phosphorylated p53 protein in RKO cells treated with 5-FU. As seen in Fig. 3A , the phosphorylated form of p53 was expressed only after 24 h of drug treatment. In contrast to the induced expression of phosphorylated p53, levels of the acetylated form of p53 remained relatively constant in cells treated with 5-FU when compared with control, non–drug-treated cells (Fig. 3B).


Figure 3
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  		Fig. 3. Effect of 5-FU on the phosphorylated and acetylated forms of p53. RKO cells were incubated with 10 µmol/L 5-FU for 24 h. Whole-cell extracts were prepared, equal amounts of cellular protein were resolved, and Western immunoblot analyses were done as described in Materials and Methods using either an anti-phosphorylated p53 rabbit polyclonal antibody (A) or an anti-acetylated lysine p53 rabbit polyclonal antibody (B). The membranes were then stripped and reprobed with an anti–ß-actin mouse monoclonal antibody.

 
Effect of 5-FU on stability of p53 protein. We next determined the effect of 5-FU treatment of the stability of p53. As seen in Fig. 4 , the half-life of p53 in control, untreated RKO cells, as determined by immunoprecipitation pulse-labeling experiments, was ~28 min. This value is consistent with prior studies that have reported the half-life of p53 to be on the order of 15 to 20 min. Treatment with 5-FU did not alter the half-life of p53 (t1/2 = 22 min) when compared with control cells. This finding suggests that the increased expression of p53, in response to exposure to 5-FU, results from newly synthesized p53 and not from enhanced stabilization of p53 protein.


Figure 4
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  		Fig. 4. Effect of 5-FU on stability of p53 in RKO cells. An immunoprecipitation, pulse-labeling experiment was done as described in Materials and Methods to determine the half-life of p53 in control, untreated (squares) and 5-FU–treated cells (diamonds). Points, mean of at least three separate experiments; bars, SE.

 
Effect of doxorubicin on p53 expression. In addition to 5-FU, we investigated the effect of the anthracycline compound doxorubicin on the induction of p53 protein in RKO cells. The cytotoxic effects associated with doxorubicin therapy are mediated by several DNA-directed mechanisms, including intercalation into DNA, inhibition of the DNA synthesis enzyme topoisomerase II, and free radical attack of DNA. Although each of these mechanisms results in DNA damage, they are presumably different than those mediated by 5-FU and the class of fluoropyrimidines. As seen in Fig. 5A , levels of p53 protein were significantly induced by up to 7-fold after treatment with 5 µmol/L doxorubicin. This concentration was selected as it represents the IC90 value, as determined by growth inhibition assay and, thus, is equitoxic to the dose of 5-FU used in the studies described above. In contrast to 5-FU, induction of p53 protein occurred at an earlier time within 4 h after doxorubicin treatment and seemed to reach maximal levels by 8 to 12 h. A Northern blot analysis revealed that levels of p53 mRNA were unchanged after doxorubicin exposure (Fig. 5B). To control for loading and integrity of RNA, we determined the expression pattern of ribosomal 28S RNA and it remained the same in control, non–drug-treated cells and in those treated with doxorubicin. The experimental data from the Western immunoblot and Northern RNA blot analyses were then pooled to form a composite graph, as presented in Fig. 5C. As with 5-FU, there was a marked discordance between the levels of p53 protein and p53 mRNA in response to treatment with doxorubicin.


Figure 5
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  		Fig. 5. A, effect of doxorubicin on expression of p53 protein. RKO cells were incubated with 5 µmol/L doxorubicin for varying periods of time (0-24 h). Whole-cell extracts were prepared, equal amounts of cellular protein were resolved, and Western immunoblot analyses were done as described in Materials and Methods. The membranes were then stripped and reprobed with an anti–ß-actin mouse monoclonal antibody. B, effect of doxorubicin on expression of p53 mRNA. RKO cells were incubated with 5 µmol/L doxorubicin for varying periods of time (0-24 h). Equal amounts (10 µg) of total cellular RNA were incubated with a biotinylated p53 antisense RNA probe and processed as described in Materials and Methods. The filter membranes were then stripped of the p53 probe and rehybridized with a human 28S antisense RNA probe to control for loading and integrity of mRNA. C, densitometric analysis of the effect of doxorubicin on expression of p53 protein and p53 mRNA levels.

 
These findings suggested that the induction of p53, in response to doxorubicin exposure, was controlled at the posttranscriptional level. However, this increased expression could be due to either translational and/or posttranslational events. As seen in Fig. 6 , levels of the phosphorylated form of p53 were significantly increased as early as 4 h after doxorubicin treatment (Fig. 6A and C). This increased expression of phosphorylated p53 reached a maximal level of 11-fold by 8 h and remained sustained for up to 24 h. The levels of acetylated p53 protein were also increased significantly by up to 4-fold in response to exposure to doxorubicin. The levels of the acetylated form continued to slowly increase during the 24-h treatment (Fig. 6B and C).


Figure 6
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  		Fig. 6. Effect of doxorubicin on the phosphorylated and acetylated forms of p53. RKO cells were incubated with 5 µmol/L doxorubicin for 24 h. Whole-cell extracts were prepared, equal amounts of cellular protein were resolved, and Western immunoblot analyses were done as described in Materials and Methods using either an anti-phosphorylated p53 rabbit polyclonal antibody (A) or an anti-acetylated lysine p53 rabbit polyclonal antibody (B). C, densitometric analysis of the time-dependent effect of doxorubicin on the expression of the phosphorylated and acetylated forms of p53.

 
Effect of doxorubicin on stability of p53 protein. Immunoprecipitation pulse-labeling studies were done to determine the half-life of p53 protein in the absence and presence of doxorubicin. As seen in Fig. 7 , the half-life of p53 in control, non–drug-treated cells was ~27 min. In contrast, in cells treated with doxorubicin, the half-life was significantly prolonged, approaching 123 min (Fig. 7). This finding suggests that the increased expression of p53 in response to doxorubicin is due to posttranslational modifications resulting in enhanced stability of the protein.


Figure 7
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  		Fig. 7. Effect of doxorubicin on biosynthesis of p53 in RKO cells. An immunoprecipitation, pulse-labeling experiment was done as described in Materials and Methods to determine the half-life of p53 in control, untreated (squares) and doxorubicin-treated cells (diamonds). Points, mean of at least three separate experiments; bars, SE.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
p53 is a critical cellular protein that plays an essential role in regulating the cell cycle and the cellular response to certain environmental and/or genotoxic stresses. Under normal growth conditions, p53 is a short-lived protein and is expressed at relatively low basal levels within the cell. In response to various cellular stresses, including exposure to DNA-damaging agents, UV and {gamma} irradiation, hypoxia, and nucleotide depletion, p53 is rapidly induced and functions as a transciptional activator. Specifically, it binds to consensus p53 binding sites expressed on a host of target genes, such as p21, GADD45, cyclin G, IGF-BP3, bcl-2, and bax, as well as a newly identified gene, p53AIP1 (4, 5, 8, 29–35). In response to genotoxic stress as observed after treatment with anticancer agents, p53 can act to arrest cell cycle progression and, in so doing, help to preserve the integrity of the cellular genome or may directly activate the process of programmed cell death.

Significant efforts have focused on characterizing the precise mechanisms by which p53 is activated by cellular stress and/or DNA damaging agents. To date, much of the work has been directed toward investigating events involving the posttranslational processing of p53. Modifications at both the N-terminal and C-terminal region of the protein have been well-documented. Whereas phosphorylation at serine 15 occurs in response to UV and {gamma} irradiation, as well as to exposure to various anticancer agents, phosphorylation at serine 389 in the COOH terminus apparently occurs only after UV irradiation and not with {gamma} irradiation (12). Acetylation at the COOH terminus is mediated by the coactivators p300, CBP, and PCAF and occurs in response to UV irradiation, resulting in activation of sequence-specific DNA binding activity (16, 17). There is also emerging evidence that the process of phosphorylation at the NH2 terminus may serve to direct acetylation at the COOH terminus (18). This finding suggests an intimate cross-talk between various domains of p53 that acts to precisely coordinate its biological activity. Recent report showed that a Mdm2 inhibitor, nutlin-3, can activate p53 binding to a level comparable with doxorubicin and 5-FU and trigger G2-M arrest compared with G1 arrest triggered by doxorubicin and 5-FU (36). This report provides further evidence that different genotoxic stress on p53 interacting protein Mdm2 can also cause unique differences in p53 activation kinetics and/or its posttranslational modification status. However, another study showed that the reduction of Mdm2 expression did not alter the response of exogenous wild-type p53 or the phosphorylation site mutants to either 5-FU or doxorubicin treatment (37). This results suggest that neither N-terminal p53 phosphorylation nor Mdm2 binding are required for p53 stabilization in response to doxorubicin or 5-FU in HCT116 cells.

In the present study, we investigated the molecular mechanisms that control the expression of p53 in response to the fluoropyrimidine 5-FU. Whereas several studies have examined the effect of various DNA-damaging agents, including UV and {gamma} irradiation, and a host of anticancer agents, including the anthracycline analogue doxorubicin, few have focused specifically on the fluoropyrimidine class of anticancer compounds. Such studies are viewed to be important as 5-FU remains an active agent for the treatment of human colorectal cancer as well as a broad spectrum of solid tumors including breast cancer, head and neck cancer, and other gastrointestinal malignancies. Our findings show that the increased expression of p53 protein after 5-FU exposure was not associated with a corresponding change in p53 mRNA levels. Although levels of phosphorylated p53 did increase after 5-FU, they did so at a later time point. Moreover, this processed form of the protein seemed to represent only a small fraction relative to the large 7-fold increase in total levels of p53 protein. Of note, 5-FU treatment did not result in increased expression of the acetylated form of the protein. Treatment with 5-FU resulted in a significant increase by up to 2.5-fold to 3-fold in the level of newly synthesized p53 with no change in the stability of the protein. Taken together, these results suggest that the induced expression of p53 in human colon cancer RKO cells treated with 5-FU is controlled at the posttranscriptional level. At time points earlier than 24 h, translational control seems to be the main regulatory process. Whereas translational regulation seems to be the dominant control mechanism at the 24-h time point, the potential role of posttranslational processing involving phosphorylation at exposure times >24 h cannot be entirely excluded.

Whereas activation of p53 expression in response to 5-FU is primarily mediated by translational control, our studies show that the induction of p53 after treatment with doxorubicin was controlled at the posttranslational level. Within a short period of time after exposure to doxorubicin, both the phosphorylated and acetylated forms of p53 were markedly increased. As a consequence of these posttranslational events, the half-life of p53 was increased significantly, by up to 6-fold to 8-fold, to 120 min. These findings are certainly consistent with those previously observed with the effect of doxorubicin treatment on p53 expression using other experimental models.

The possibility for translational regulation as a mechanism to control p53 expression in response to genotoxic stress has been suggested previously by other investigators (19, 20). Kastan et al. (19) observed that exposure of ML-1 myeloblastic leukemia cells to nonlethal doses of the DNA-damaging agents, {gamma} irradiation or actinomycin-D resulted in significant increases in p53 expression. The levels of p53 mRNA did not change with exposure to either of these genotoxic agents, and concomitant treatment with the protein synthesis inhibitor cycloheximide completely inhibited synthesis of new p53 protein. Of note, cells expressing mutant or inactivated p53 were unable to express increased levels of protein in response to DNA-damaging agents, providing evidence that this induction is dependent on the presence of wild-type p53. These findings suggested a posttranscriptional process with translational regulation being a likely control mechanism. Fu and Benchimol (22, 23) observed that the increase in p53 protein, in response to ionizing radiation, was accompanied by a large increase in the association of p53 mRNA with higher molecular weight polysomes without any appreciable change in the level of p53 mRNA. This work provided the first direct evidence for the role of translational regulation in controlling p53 expression after treatment with a DNA-damaging agent.

Recent studies have documented that the expression of p53 is controlled by a translational autoregulatory feedback loop in which p53 protein interacts directly with a target sequence on its cognate p53 mRNA. In murine systems, p53 protein interacts with the 5' untranslated region of the p53 mRNA, whereas the cis-acting element on the human p53 mRNA has been localized to the 3' untranslated region. The in vivo biological relevance of the p53 protein–p53 mRNA interaction has been further supported by the isolation of p53 protein–p53 mRNA complexes from whole-cell extracts of rat embryonic fibroblasts (38). At present, significant efforts are directed toward characterizing the molecular elements underlying this RNA-protein interaction, with particular focus on identifying the specific RNA binding domain(s) on p53 protein.

In addition to the process of p53 translational autoregulation, p53 mRNA translation can be controlled by the folate-dependent enzyme TS. In this setting, our laboratory has shown that TS binds to a sequence in the protein-coding region of p53 mRNA and, in so doing, functions as a translational repressor (24, 25). The potential in vivo relevance of the TS protein–p53 mRNA interaction is supported by the isolation of RNP complexes composed of TS protein and p53 mRNA from H630 human colon cancer cells. Moreover, the biological significance of the TS protein–p53 mRNA interaction has been confirmed by transfection studies in which constitutive overexpression of human His-Tagged TS protein in human colon cancer HCT-C cells with a functionally inactive TS results in nearly complete suppression of p53 biosynthesis (25). A series of detailed experiments has shown that the reduced expression of p53 resulted from a decreased translational efficiency of p53 mRNA. Cells overexpressing TS with reduced levels of p53 were significantly impaired in their ability to arrest at both the G1 or G2 checkpoints in response to {gamma} irradiation and/or various anticancer agents. Recently, our group has done a comprehensive gene expression profile and identified some 150 genes that are affected by TS overexpression, as well as many novel posttranscriptionally regulated genes affected by 5-FU treatment (39). Together, these findings suggest that the TS protein–p53 mRNA interaction may play a critical role in the cellular response to genotoxic stress.

In this study, we show that the increased expression of p53 in response to 5-FU treatment is regulated at the translational level. There are several potential advantages offered by this control mechanism. Immediacy and rapidity represent a clear advantage of translational control over transcriptional and/or other nuclear regulatory mechanisms. Because translation involves the last critical step in protein biosynthesis, there is no delay in implementing rapid changes. Second, this control mechanism is readily reversible and is therefore economical in terms of energy requirements. This feature represents a particularly significant advantage in malignant cells that have been exposed to a cytotoxic stress. Third, translational control avoids the need for transcribing RNA from a template DNA that has been damaged by a cytotoxic agent and, in so doing, would help to preserve the integrity of the cell. Finally this process allows the expression of a given gene to be more precisely fine-tuned.

At present, translation of p53 mRNA is controlled by binding with its own protein end-product p53 or with the folate-dependent enzyme TS. It is conceivable that treatment with 5-FU could, in some way, abrogate the normal p53 mRNA–p53 protein or p53 mRNA–TS protein interactions, thereby leading to enhanced p53 mRNA translation. A second possibility is that 5-FU or one of its metabolites could alter the expression and/or function of certain key cofactors or other cellular proteins required for these p53 mRNA–protein interactions. Further studies are now under way to more precisely characterize the molecular mechanisms involved in the 5-FU–mediated induction of p53 mRNA translation.

In conclusion, we show that the induction of p53, in response to genotoxic stress, is controlled by both translational and posttranslational mechanisms with unique kinetics. The specific mechanism controlling the increased expression of p53 is dependent on the specific anticancer agent under investigation. This work expands our current understanding of the molecular mechanisms that control p53 expression and provides further evidence for the central role of translational and posttranslational regulation in the control of cellular gene expression. Further studies are required to more carefully elucidate the essential molecular elements that signal which of these regulatory pathways are activated in the cellular response to genotoxic stress.


   Acknowledgments
 
We thank Drs. Xiukun Lin, Jun Liu, Ashwin Gollerkeri, and Tian-Men Chen for helpful discussions and for the review of the manuscript.


   Footnotes
 
Grant support: National Cancer Institute National Research Scholar award CA81690 (J. Ju), Department of Veterans Affairs VA Merit Review award (E. Chu), and National Cancer Institute grants CA16359 and CA75712 (E. Chu).

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.

Received 12/ 7/06; revised 4/10/07; accepted 5/ 2/07.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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