Deoxycholic acid suppresses p53 by stimulating proteasome-mediated p53 protein degradation
Dianhua Qiao,
Supriya V. Gaitonde,
Wenqing Qi and
Jesse D. Martinez,1
Arizona Cancer Center, Department of Radiation Oncology, University of Arizona, Tucson, AZ 85724, USA
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Abstract
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Bile acids, principally deoxycholic acid (DCA), have been implicated in the promotion of colon tumorigenesis in both animals and humans. Increasing evidence suggests that bile acids may exert their tumor promoting activity by modulating intracellular signaling and altering gene expression. In this study we have investigated the effect of bile acids on the tumor suppressor p53 using the human colon tumor cell line HCT116, which retains the wild-type p53 gene and functional p53 signaling in response to DNA damage. We found that exposure of the cells to elevated concentrations of DCA suppressed accumulation of p53 protein as well as p53 transactivation and impaired the p53 response of the cells to DNA damaging agents, such as ionizing radiation. Neither ursodeoxycholic acid, a putative chemopreventive agent, nor cholic acid, which is biologically inert, had any effect on p53 protein level and transactivation activity. Further examination revealed that instead of inhibition, DCA induced p53 mRNA in a dose-dependent manner, indicating that the inhibitory effect of DCA on p53 protein is mediated by a post-transcriptional mechanism. Both lactacystin, a specific inhibitor of the 26S proteasome, and leptomycin B, a specific inhibitor of the nuclear export protein CRM1, could block the effect that DCA had on p53 protein levels, suggesting that DCA suppressed p53 by stimulating the process of proteasome-mediated degradation of p53. Significantly, blocking extracellular signal-regulated kinase (ERK) signaling, but not protein kinase C (PKC), blunted suppression by DCA of p53 protein levels and transactivation activity, suggesting that DCA suppressed p53, in part, by stimulating the ERK signaling pathway. Both ERK and PKC signaling have been previously demonstrated to be stimulated by DCA. These results suggest a novel signaling mechanism of bile acids that may play an important role in colon tumor promotion mediated by bile acids.
Abbreviations: AOM, azoxymethane; AP-1, activator protein-1; CA, cholic acid; DCA, deoxycholic acid; DMEM, Dulbecco's modified Eagle's medium; GADPH, glycerddehyde 3-phosphate dehydrogenase; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; UDCA, ursodeoxycholic acid.PKC, protein kinase C; UDCA, ursodeoxycholic acid.
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Introduction
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Colorectal cancer remains a major health problem and ranks as the second most common cause of cancer deaths in Western countries. Epidemiological studies have shown that elevated fecal bile acids are associated with an increased risk of colon cancer. For example, Western populations that consume high fat diets have, on average, elevated fecal bile acid concentrations and an increased risk for developing colon cancer compared with Eastern populations that consume diets that are lower in fat (1). In addition, patients with colonic adenomas and carcinomas also tend to have elevated concentrations of bile acids in their blood and stool (24). However, the most compelling evidence for a causal link between fecal bile acids and increased colonic carcinogenesis comes from animal model studies. Rats treated with azoxymethane (AOM) and fed a diet supplemented with the bile acid deoxycholic acid (DCA) develop more tumors than do animals treated with AOM alone (57). Furthermore, in an elegant series of experiments Morvay and co-workers demonstrated directly by surgically altering the flow of bile acids in the colon that the presence or absence of bile acids is a strong determinant of tumor incidence (8). However, bile acids by themselves cannot induce tumors (9). Hence, bile acids, principally DCA, are tumor promoters that may play an important role in the pathogenesis of colon cancer.
The mechanism by which DCA or other bile acids function is still unclear. However, recent studies have shown that bile acids can affect intracellular signaling and gene expression, which may ultimately lead to alterations in cell growth and tumorigenesis. A variety of growth regulatory genes, such as cyclooxygenase-2 (10), GADD153 (11) and c-myc (12,13), and transcription factors, such as activator protein-1 (AP-1) (14) and NF-
B (15), are reportedly activated by bile acids. Furthermore, it has been demonstrated that bile acids can stimulate intracellular signaling cascades including protein kinase C (PKC) and mitogen-activated protein kinases (MAPK) (14,16). PKC and MAPK are two groups of important signaling mediators that transduce various extra- or intracellular signals to gene regulation, cell growth control or changes in tumorigenesis (17,18). Several studies have shown that bile acids may induce apoptosis and activate some genes, including cyclooxygenase-2 and GADD153, via a signaling cascade that includes PKC, MAPK and AP-1 (10,14). Bile acid-induced apoptosis has been suggested to play an important role in bile acid promotion of colon tumorigenesis by providing selection for tumorigenic cells in the colon (9). This, combined with the ability of bile acids to stimulate proto-oncogenes, such as cyclooxygenase-2, c-myc and AP-1, suggests that bile acids may exert their tumor promoting activity by modulating intracellular signaling and gene expression, which consequently change cell growth and tumorigenesis.
Bile acids act as tumor promoters rather than complete carcinogens (9). In view of the fact that cancer is a genetic disease and accumulation of genomic mutations plays a determinative role in tumorigenesis, it is possible that bile acid signaling may interfere with the function of tumor suppressor genes involved in maintaining genome integrity in response to DNA damage. Among the most important tumor suppressor genes is p53, which acts as a transcription factor and is responsible for monitoring DNA damage and suppressing tumorigenesis in mammalian cells (19). With this in mind, we examined whether bile acids could influence p53 activity. Our results showed that the tumor-promoting bile acid DCA could reduce p53 protein levels as well as transactivation activity. Importantly, DCA was able to suppress the accumulation of p53 in response to DNA-damaging agents such as ionizing radiation. These results may provide an important insight into the molecular mechanisms by which bile acids promote colon tumorigenesis.
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Materials and methods
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Plasmids and reagents
pG13-luc, a luciferase reporter driven by a promoter sequence containing multiple copies of a p53 binding site, and pC53-SN3, a human p53 cDNA clone in the pCMV-Bam-neo vector, were kindly provided by Dr B.Vogelstein (20). pRL-CMV, a constitutive expression construct for renilla luciferase, was from Promega (Madison, WI). pTZ-GAP contains a 0.75 kb human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA fragment (21). LipofectAMINE and TRIzol Reagent were from Gibco BRL (Gaithersburg, MD). The sodium salts of DCA, ursodeoxycholic acid (UDCA) and cholic acid (CA) were from Sigma (St Louis, MO) and were maintained as 100 mM stock solutions in water. PD 98059 (2'-amino-3'-methoxyflavone), bisindolylmaleimide I, lactacystin and leptomycin B were from Calbiochem (La Jolla, CA) and were maintained as stock solutions in dimethyl sulfoxide.
Cell culture, treatment and transfection
HCT116 (ATCC, MD), a cell line derived from an adenocarcinoma patient with Lynch's syndrome, and normal human fibroblasts (from Steve St Jeor, University of Nevada, Reno, NV) were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL) with 10% fetal bovine serum, 2 mM L-glutamine and 100 units/ml penicillin/streptomycin at 37°C in an incubator containing 5% CO2. All experiments for detection of p53 protein and mRNA were conducted with cells grown to 7080% confluency. Treatment of the cells with chemical inhibitors was performed by pretreating the cells with the inhibitors for 30 min prior to addition of bile acids. The inhibitors persisted in the medium until the cells were harvested.
-Irradiation of the cells was conducted using 60Co immediately after addition of DCA.
Transient transfection was conducted by cationic lipid-mediated DNA transfection using LipofectAMINE. In brief, 3x105 HCT116 cells were seeded in 35 mm dish and the cells cultured until they reached a confluency of 5070%. The cells were washed with serum/antibiotic-free DMEM medium prior to transfection. Aliquots of 0.5 µg each of plasmids pG13-luc and pRL-CMV purified with Qiagen tips (Qiagen, Valencia, CA) and 3 µl of LipofectAMINE reagent diluted in 100 µl of serum/antibiotic-free DMEM medium were mixed gently and incubated at room temperature for 45 min. After adding 0.8 ml of serum/antibiotic-free DMEM medium the lipidDNA complex was placed onto the cells. Transfection was performed at 37°C in an incubator under 5% CO2 for 5 h. After transfection the lipidDNA complex was replaced by fresh culture medium with or without the reagents as indicated for designated treatments of the cells. The cells were then cultured for another 12 h before harvest.
Immunoblotting and immunoprecipitation
Cells were lysed in 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerol phosphate, 1 mM sodium vanadate and 10 mM sodium fluoride. Aliquots of 50 µg total cellular protein were subjected to SDSPAGE in 10% gels. Western blot analyses for p53 or
-tubulin were performed using mouse monoclonal antibody against human p53 protein (DO1; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit polyclonal antibodies against human
-tubulin (Santa Cruz). Horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG antibodies (Santa Cruz) and Luminol ECL reagents (Amersham Life Science, Little Chalfont, UK) were used to visualize the specific immunocomplex by autoradiography. The relative amount of p53 protein detected was measured as integrated density by scanning the autoradiograph on a Stratagene Eagle Eye II (Stratagene, La Jolla, CA). For immunoprecipitation the cell lysates were incubated with protein Aagarose beads (Gibco BRL) at 4°C with rotation for 30 min. After microcentrifugation the protein concentration of the supernatant was determined. An aliquot of 500 µg total protein was mixed with 200 µl of the PAb421 monoclonal antibody against p53 and 60 µl of 50:50 protein Aagarose beads and incubated at 4°C with rotation overnight. The beads were then washed twice with the lysis buffer and boiled for 5 min after adding 60 µl of 2x SDSPAGE sample buffer (100 mM Tris, pH 6.8, 200 mM dithiothreitol, 4% SDS, 20% glycerol, 0.001% bromophenol blue).
RNA isolation and northern blot analysis
Total cellular RNA was isolated from treated and untreated HCT116 cells using TRIzol Reagent according to the manufacturer's instructions. Aliquots of 20 µg RNA were fractionated on formaldehydeagarose gels and transferred to Biotrans nylon membranes (ICN Biomedicals, Aurora, OH). The human p53 and GAPDH cDNA fragments were labeled with [
-32P]dATP by nick translation and hybridized separately with the RNA blot at 68°C in 7% SDS, 1% bovine serum albumin, 100 µg/ml salmon sperm DNA and 0.25 M sodium phosphate, pH 7.2. The membrane was washed after hybridization for 30 min twice in 1x SSC, 0.1% SDS at 68°C.
Luciferase assay
The firefly and renilla luciferase activities in the cells transiently transfected with both pG13-luc and pRL-CMV were measured using a Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. A Monolight 3010 luminometer (Analytical Luminescence Laboratory, San Diego, CA) was used. The firefly luciferase activity produced by pG13-luc was normalized relative to renilla luciferase activity produced by pRL-CMV and expressed as a ratio.
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Results
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DCA suppresses the intracellular level of p53 protein as well as transactivation
Induction of p53 function in response to DNA damage and certain cellular stresses is initiated by the accumulation of p53 protein in the cells (22). To investigate the effect of bile acid signaling on p53 we used the human colon cancer cell line HCT116, which contains a detectable level of wild-type p53 and possesses an intact G1/S checkpoint in response to DNA damage, indicating functional p53 signaling in these cells (23). We treated the cells with different concentrations of DCA or 200 µM UDCA, a chemopreventive bile acid, or CA, a biologically null bile acid, for 12 h and the p53 protein level was examined by immunoblot analysis using an antibody against total p53 protein. The same blot was also probed with an antibody against human
-tubulin to control for variations in loading. The results showed that at 200 µM the tumor-promoting bile acid DCA caused a considerable decrease (~40%) in p53 protein level (Figure 1
). However, neither UDCA nor CA had any effect on levels of p53 in HCT116 cells. Notably, this effect was dose dependent and occurred only at elevated concentrations of DCA. Statistical analysis of 12 independent experiments showed that DCA suppression of p53 was statistically significant (P < 0.001, Student's t-test) and at 12 h p53 levels were reduced by 200 µM DCA to an average of 57.8% relative to controls, with a SD of 8.4%. In addition, the observed reduction in p53 was not due to apoptosis since at
200 µM DCA-induced apoptosis was only 24% after 12 h. DCA-induced cell death has been previously characterized as typical apoptosis (9) and it was detected by fluorescent microscopy using the fluorescent dyes acridine orange and ethidium bromide (41). Moreover, in contrast to the inhibitory effect of DCA on p53, previous studies in HCT116 cells showed that DCA at
200 µM did not affect or even increased the cellular levels of some other proteins (14,30). Hence, exposure to DCA specifically suppressed p53 protein levels in HCT116 cells and this reduction was not due to cytotoxicity.
p53 acts as a transcription factor and exerts its growth inhibitory effect, i.e. inducing cell cycle arrest or apoptosis, mostly by transactivating downstream target genes. In fact, it has been argued that it is not the total amount of p53 protein that is critical, but rather the amount of activity (19). Thus, we examined whether DCA suppressed the transcriptional activity of p53. For this purpose HCT116 cells were transiently transfected with the p53luciferase reporter pG13-luc and the constitutive renilla luciferase construct pRL-CMV. Immediately after transfection the cells were exposed to different concentrations of DCA or 200 µM UDCA or CA for 12 h. The luciferase activity produced by pG13-luc was determined and normalized to the renilla luciferase activity. As shown in Figure 2
, at concentrations >150 µM DCA caused a dose-dependent decrease in p53 transactivation activity. However, consistent with the p53 protein levels observed in immunoblot analyses (Figure 1
), both UDCA and CA had no effect on p53 transactivation activity. Furthermore, the expression levels of p53 target genes, such as mdm2, were determined to confirm the inhibitory effect of DCA on p53 transcriptional activity. As shown in Figure 7A
, both p53 and MDM2 protein levels were consistently reduced by DCA. These results taken together indicate that DCA can suppress both p53 protein levels and transactivation.

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Fig. 2. Effect of bile acids on p53 transactivation. HCT116 cells were transiently transfected with the p53luciferase reporter pG13-luc and the constitutive renilla luciferase construct pRL-CMV. The transfected cells were treated with increasing concentrations of DCA (A) or with three different bile acids each at 200 µM (B) as indicated for 12 h. The cells were harvested and luciferase activities examined. The luciferase activity produced by pG13-luc was normalized relative to renilla luciferase activity produced by pRL-CMV and expressed as a ratio. Each bar represents the mean ± SE of two independent experiments.
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Fig. 7. Effect of DCA on the p53MDM2 interaction and p53 phosphorylation at Ser15. (A) HCT116 cells were treated with 200 µM DCA for different time periods as indicated and total cellular extracts prepared. Samples of 50 µg total protein were subject to SDSPAGE and western blot analyses were performed using mouse monoclonal antibodies against human p53 (DO1; Santa Cruz) or MDM2 (Santa Cruz). Samples of 500 µg total protein were immunoprecipitated with the PAb421 monoclonal antibody against human p53. Western blot analyses were performed with the immunoprecipitates using mouse monoclonal antibody against human MDM2 (Santa Cruz). (B) HCT116 cells were pretreated with 40 µM PD 98059 for 30 min and then treated with 200 µM DCA or 5 Gy -radiation for different time periods, as indicated, in the presence of PD 98059. Total cellular extracts were prepared and 50 µg total protein were used for immunoblot analyses using monoclonal antibodies against human Ser15 phospho-p53 (Santa Cruz) or human total p53 (DO1; Santa Cruz).
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DCA increases p53 transcript
Like other genes, p53 regulation may occur at multiple levels, including transcription, post-transcriptional regulation of mRNA, translation and post-translational modification (24). In order to clarify whether DCA suppresses p53 transcription we examined the steady-state levels of p53 transcript by northern blot analysis. HCT116 cells were treated with different concentrations of DCA or 200 µM UDCA or CA for 12 h and northern blot analyses conducted using radiolabeled p53 and GAPDH cDNA probes. Surprisingly, exposure to DCA resulted in a dose-dependent induction of p53 mRNA (Figure 3
). Neither UDCA nor CA affected p53 mRNA levels (Figure 3C and D
). Although it is not clear whether this mRNA induction was accomplished by stimulating gene transcription or by stablizing the mRNA itself, these results clearly indicated that DCA suppression of p53 did not occur by reducing the transcript levels of p53.

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Fig. 3. Effect of bile acids on p53 mRNA levels. HCT116 cells were treated with different concentrations of DCA (A and B) or with three different bile acids at a final concentration of 200 µM (C and D) as indicated for 12 h. Total RNAs were isolated and northern blot analyses performed using a radiolabeled human p53 cDNA probe. The filters were stripped and re-probed with radiolabeled human GAPDH cDNA. Each hybridization was quantitated on a PhosphorImager (Molecular Dynamics) and expression of p53 mRNA was normalized for loading variations using GAPDH and expressed relative to that seen in untreated cells (B and D).
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DCA mediates p53 down-regulation by stimulating the proteasomal degradation of p53
p53 protein level is normally rigorously controlled under both normal and stressful conditions by a fine tuned ubiquitination-mediated proteasomal degradation (25). To determine whether DCA mediated p53 reduction by stimulating proteasomal degradation of p53 a specific inhibitor of proteasomal activity, lactacystin (26), was used and its effect on DCA-mediated p53 reduction was examined. The results showed that lactacystin can stabilize p53 protein both in the absence and presence of DCA and at 5 µM the inhibitor completely blocked the effect of DCA on p53 protein levels (Figure 4
). Previous studies have shown that export of p53 from the nucleus to the cytoplasm is the essential step for p53 to undergo ubiquitination-mediated proteasomal degradation (25). With this in mind we treated the cells with leptomycin B (27), a specific inhibitor of the nuclear export protein CRM1, and the effect of DCA on p53 protein levels was examined. The results showed that blocking p53 nuclear export completely abolished the DCA-mediated reduction in p53 protein (Figure 5
). These taken together suggest that DCA-mediated suppression of p53 is accomplished by stimulating the process of proteasomal degradation of the protein.
Extracellular signal-regulated kinase (ERK) partially mediates DCA-induced p53 degradation
Increasing evidence suggests that bile acids affect gene expression and cell growth as well as tumorigenesis by modulating intracellular signaling. Extensive studies both in vitro and in vivo have shown that bile acids can stimulate PKC in colon cells and this may play an important role in bile acid-mediated cellular processes (16,28,29). In addition, we previously showed that DCA could activate ERKs (30). There is evidence suggesting that both PKC and ERK may participate in negative regulation of p53 in cells (3234). Hence, it was of interest to determine whether DCA down-regulated p53 via a signaling pathway which involves PKC or ERK. For this purpose HCT116 cells were treated with different concentrations of bisindolylmaleimide I (Bis), a highly selective and potent PKC inhibitor which competes for the ATP-binding site of PKC isoenzymes (35), or PD 98059, a specific inhibitor of MAPK/ERK kinase (MEK) (36), and their effect on DCA-mediated p53 protein reduction was examined. The results showed that Bis had no effect on suppression by DCA of p53 protein, even at a much higher concentration, such as 10 µM (data not shown). According to previous studies with Bis, 10 µM is far beyond the concentrations at which Bis can effectively suppress cellular PKC activity (35,42). However, inhibition of ERK by PD 98059 increased the p53 protein levels in both untreated and DCA-treated cells in a dose-dependent manner (Figure 6
). Notably, at either 20 or 40 µM PD 98059 increased p53 protein in cells treated with DCA to a level comparable to that observed in untreated cells. Further examination of the transactivation activity of p53 in the cells showed that PD 98059 had a similar effect on p53 transactivation as on protein levels (Figure 6C
). Our previous studies have demonstrated that at concentrations >20 µM PD 98059 can effectively eliminate both basal and DCA-induced ERK activity in HCT116 cells (14). Despite the fact that inhibition of ERK did not completely block DCA-mediated suppression of p53 protein level or transactivation (Figure 6
), these results suggest that ERK, but not PKC, may contribute to DCA-induced down-regulation of p53.
The effect of DCA on p53 phosphorylation and p53MDM2 interaction
Interaction of MDM2 with the N-terminal transactivation domain of p53 has been shown to be the critical event upstream of p53 nuclear export, ubiquitination and proteasomal degradation (38). Enhancement of the p53MDM2 association could result in more rapid degradation of p53. To test whether DCA accelerates p53 degradation by increasing the p53MDM2 interaction HCT116 cells were treated with 200 µM DCA for different time periods and MDM2 protein levels in both total cellular extracts and p53MDM2 complexes immunoprecipitated with the anti-p53 monoclonal antibody PAb421 were determined. As shown in Figure 7A
, both MDM2 protein levels in total cellular extracts and p53MDM2 complexes were consistently reduced by DCA, as seen for p53 levels, suggesting that DCA treatment did not increase p53MDM2 interaction while p53 was degraded.
Previous studies have shown that the p53MDM2 interaction can be directly regulated by phosphorylation of serine/threonine residues located in the N-terminus of p53, in particular Ser15 (31). To investigate the impact of DCA on p53 phosphorylation and confirm the observation that DCA did not affect p53MDM2 interaction HCT116 cells were treated with DCA for different time periods and the Ser15 phosphorylation status of p53 was determined by immunoblotting using a phospho-specific antibody. As a positive control for Ser15 phosphorylation of p53 the cells were also treated with ionizing
-radiation. It has been reported that ERK-mediated Ser15 phosphorylation of p53 is involved in cisplatin-induced p53 accumulation (43). However, our observations in this study showed that ERK activity was associated with p53 degradation in HCT116 cells (Figure 6
). In order to further understand the role of ERK in DCA-mediated p53 degradation the cells were treated with PD 98059 and its effect on Ser15 phosphorylation of p53 was examined. As shown in Figure 7B
, ionizing
-radiation induced phosphorylation of p53 at Ser15 as described previously (44). Neither DCA nor PD 98059 showed any inhibitory effect on Ser15 phospho-p53 levels in the cells, although total p53 levels were affected by both agents. The inability of both DCA and PD 98059 to down-regulate Ser15 phosphorylation in p53 is consistent with the observations that DCA did not increase p53MDM2 interaction and that DCA-stimulated ERK suppressed p53.
DCA suppresses ionizing radiation-induced p53 activation
Colon epithelium is constantly exposed to various stressful environmental factors derived from the diet, including those that can induce DNA damage. Suppression of the p53 response to these harmful luminal components could lower the ability of colon cells to eliminate tumorous transformation. To investigate the potential of DCA in this effect HCT116 cells were treated with 5 Gy
-radiation in the presence or absence of 200 µM DCA. After 12 h the cells were harvested and p53 protein levels determined by immunobloting. p53 protein was induced when cells were exposed to
-radiation whereas this induction was apparently suppressed by exposure to DCA (Figure 8A
). Transient transfection experiments with plasmids pG13-luc and pRL-CMV were also conducted and the results showed that DCA can inhibit
-radiation-induced p53 transactivation (Figure 8B
). This inhibitory effect of DCA on p53 protein was further confirmed in the normal human fibroblast cell line HEL (Figure 8C
).
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Discussion
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Epidemiological and animal studies have demonstrated that bile acids, principally DCA, are endogenous colon tumor promoters (18). Increasing evidence suggests that bile acids may exert their tumor-promoting activity by affecting intracellular signaling, which leads to alterations in gene expression and cell growth as well as tumorigenesis. Understanding the signaling mechanisms associated with bile acid tumor-promoting activity will be helpful for the development of successful chemoprevention strategies for colon cancer. In this study we have investigated the influence of different bile acids on the tumor suppressor p53. We have shown, for the first time, that DCA, but not UDCA nor CA, can suppress p53 protein levels and transactivation activity. With the correlation between the tumor-promoting activity of DCA and its capacity to interfere with p53 it seems that repression of p53 signaling may be associated with bile acid-mediated tumor promotion.
Both protein levels and transactivation activity of p53 were accordingly reduced in response to DCA treatment (Figures 1 and 2
), suggesting that suppression of p53 activity by DCA is essentially due to a decrease in p53 protein levels. In an attempt to determine whether DCA suppressed p53 protein levels by reducing p53 mRNA amounts we were surprised to find that DCA induced p53 mRNA in a dose-dependent manner (Figure 3
). This is contradictory to the effect of DCA on p53 protein levels, however, it clearly indicated that DCA suppression of p53 protein levels occurred by a mechanism that may involve down-regulation of p53 mRNA translation or p53 protein stability. The induction of p53 mRNA by DCA was probably caused by increasing p53 gene transcription, although we cannot exclude the possibility that DCA may stabilize p53 mRNA. Our previous studies have shown that DCA can activate AP-1 in HCT116 cells, even at low concentrations such as 50 µM (14). In addition, it has been reported by others that DCA could stimulate the transcription factors NF-
B and c-Myc (12,13). Binding sites for all of these transcription factors have been found in the p53 promoter and may account for the increased p53 mRNA levels (37). However, the significance of this mRNA induction in colon tumorigenesis remains to be determined.
p53 protein is normally targeted for degradation by the 26S proteasome following ubiquitination, which has been demonstrated to be the most common mechanism by which p53 is controlled (25). In the present study blocking proteasome-mediated protein degradation with a specific inhibitor, lactacystin (26), resulted in accumulation of p53 and complete elimination of the inhibitory effect of DCA on p53 protein levels (Figure 4
). These results suggest that in HCT116 cells p53 protein is constantly subjected to proteasomal degradation and that DCA suppresses p53 by stimulating proteasome-mediated degradation. This notion was further supported by the observation that inhibition of p53 degradation by leptomycin B (27), a specific inhibitor of the nuclear export protein CRM1, abolished the DCA-mediated reduction in p53 protein (Figure 5
).
At present MDM2 is well established as the principle regulator of p53 ubiquitination and degradation. MDM2 binding within the transactivation domain of p53 leads to repression of p53 transactivation, exclusion of p53 protein from the nucleus and localization in the cytoplasm, where p53 protein is further ubiquitinated by MDM2 and other ubiquitin ligases and consequently targeted for degradation by the 26S proteasome (38). Our results in this study showed no increase in the p53MDM2 interaction in DCA-treated cells, suggesting that DCA may not accelerate p53 degradation by strengthening the p53MDM2 interaction (Figure 7A
). This notion is consistent with the observation that DCA treatment did not affect Ser15 phospho-p53 levels in the cells despite the total p53 levels being reduced (Figure 7B
). The Ser15 phosphorylation site is juxtaposed to the MDM2-binding site (residues 1823) and it has been suggested that phosphorylation of p53 at Ser15 can block its interaction with MDM2 (31). Nevertheless, several other processes that are involved in MDM2-mediated p53 degradation, including nuclear export of p53MDM2 and ubiquitination and proteasomal degradation of p53, might be targeted by DCA.
Post-translational modification, including phosphorylation and acetylation, of p53 and MDM2, as well as other relevant regulatory or functional proteins, plays critical roles in the regulation of p53 stability (31). With the ability of bile acids to affect intracellualr signaling it is conceivable that bile acids may influence p53 by altering post-translational modification. In this study we have shown that inhibition of ERK led to stabilization of p53 protein as well as an increase in transcriptional activity (Figure 6
). It has been demonstrated in our previous studies that ERK is strongly activated by DCA (14). These taken together suggest that DCA may suppress p53 through activating ERK. This notion is supported by the observations that inhibition of ERK could suppress bryostatin-1-induced p53 degradation in NB4 leukemia cells (32) and that Ras regulation of sympathetic neuron survival was mediated by activation of both the PI3-K- and MEK-signaling cascades, which in turn suppressed a pro-apoptotic p53 pathway (33). However, ERK activation can only account for part of the inhibitory effect that DCA had on p53 because blocking ERK activation did not completely eliminate suppression of p53 by DCA. Inhibition of PKC did not alter the effect of DCA on p53 protein, suggesting that DCA-stimulated PKC signaling may not be associated with DCA-induced p53 degradation (data not shown). The mechanism by which ERK inhibits p53 remains unknown. Our investigation has revealed that disturbance of DCA-induced ERK does not affect the phosphorylation status of p53 at Ser15 (Figure 7B
). More studies will be needed to determine other signaling mechanisms that are stimulated by DCA and contribute to DCA suppression of p53 in addition to ERK.
Colon cancer is a typical multistage cancer and cancer genetic studies have suggested that p53 mutation is a determinative step in colonic carcinogenesis (39). Bile acids lack the ability to initiate tumorigenesis basically due to their inability to directly induce DNA damage. The general consensus at the moment is that bile acids promote colon tumorigenesis by affecting intracellular signaling. In this study we have pointed out a direct link between tumor-promoting bile acids and the tumor suppressor gene p53. More significantly, DCA can inhibit p53 activation mediated by DNA-damaging agents, such as ionizing
-radiation (Figure 8
). p53 plays critical roles in maintenance of genome integrity and suppression of tumorigenesis by sensing DNA damage and inducing cell cycle arrest and apoptosis as well as directly participating in DNA repair (40). Suppression of the p53 response to genotoxic compounds present in the colon could enhance mutagenesis and, ultimately, lead to increased cancer risk.
In conclusion, we have shown in this study that the tumor-promoting bile acid DCA can negatively affect p53 through stimulating proteasome-mediated p53 protein degradation and that this may be partially mediated by DCA-stimulated ERK signaling. Our results suggest that bile acids may promote colon tumorigenesis by suppressing p53 function by altering intracellular signaling.
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Notes
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1 To whom correspondence should be addressed Email: jmartinez{at}azcc.arizona.edu 
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Acknowledgments
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We thank Dr B.Vogelstein for providing the pG13-luc and pC53-SN3 plasmids. This work was supported by National Institutes of Health Grant CA-72008 and Cancer Center Core Grant CA-23074.
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References
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---|
-
Armstrong,B. and Doll,R. (1975) Environmental factors and cancer incidence and mortality in different countries, with special reference to dietary practices. Int. J. Cancer, 15, 617631.[ISI][Medline]
-
Bayerdorffer,E., Mannes,G.A., Richter,W.O., Ochsenkuhn,T., Wiebecke,B., Kopcke,W. and Paumgartner,G. (1993) Increased serum deoxycholic acid levels in men with colorectal adenomas [see comments]. Gastroenterology, 104, 145151.[ISI][Medline]
-
Kishida,T., Taguchi,F., Feng,L., Tatsuguchi,A., Sato,J., Fujimori,S., Tachikawa,H., Tamagawa,Y., Yoshida,Y. and Kobayashi,M. (1997) Analysis of bile acids in colon residual liquid or fecal material in patients with colorectal neoplasia and control subjects. J. Gastroenterol., 32, 306311.[ISI][Medline]
-
Reddy,B.S. and Wynder,E.L. (1977) Metabolic epidemiology of colon cancer. Fecal bile acids and neutral sterols in colon cancer patients and patients with adenomatous polyps. Cancer, 39, 25332539.[ISI][Medline]
-
Reddy,B.S., Watanabe,K., Weisburger,J.H. and Wynder,E.L. (1977) Promoting effect of bile acids in colon carcinogenesis in germ-free and conventional F344 rats. Cancer Res., 37, 32383242.[ISI][Medline]
-
Sutherland,L.A. and Bird,R.P. (1994) The effect of chenodeoxycholic acid on the development of aberrant crypt foci in the rat colon. Cancer Lett., 76, 101107.[ISI][Medline]
-
Narisawa,T., Magadia,N.E., Weisburger,J.H. and Wynder,E.L. (1974) Promoting effect of bile acids on colon carcinogenesis after intrarectal instillation of N-methyl-N'-nitro-N-nitrosoguanidine in rats. J. Natl Cancer Inst., 53, 10931097.[ISI][Medline]
-
Morvay,K., Szentleleki,K., Torok,G., Pinter,A., Borzsonyi,M. and Nawroth,R. (1989) Effect of change of fecal bile acid excretion achieved by operative procedures on 1,2-dimethylhydrazine-induced colon cancer in rats. Dis. Colon Rectum, 32, 860863.[ISI][Medline]
-
Martinez,J.D., Stratagoules,E.D., LaRue,J.M., Powell,A.A., Gause,P.R., Craven,M.T., Payne,C.M., Powell,M.B., Gerner,E.W. and Earnest,D.L. (1998) Different bile acids exhibit distinct biological effects: the tumor promoter deoxycholic acid induces apoptosis and the chemopreventive agent ursodeoxycholic acid inhibits cell proliferation. Nutr. Cancer, 31, 111118.[ISI][Medline]
-
Zhang,F., Subbaramaiah,K., Altorki,N. and Dannenberg,A.J. (1998) Dihydroxy bile acids activate the transcription of cyclooxygenase-2. J. Biol. Chem., 273, 24242428.[Abstract/Free Full Text]
-
Zheng,Z., Bernstein,H., Bernstein,C., Payne,C.M., Martinez,J.D. and Gerner,E.W. (1996) Bile acid activation of the gadd153 promoter and of p53-independent apoptosis: relevance to colon cancer. Cell Death Differ., 3, 407414.[ISI]
-
Takai,Y., Kaibuchi,K., Tsuda,T., Yamashita,T., Kikuchi,A., Tanimoto,T. and Hoshijima,M. (1986) Possible modes of action of growth factors and tumor promoters in the activation of the c-myc gene in Swiss 3T3 fibroblasts (in Japanese). Gan To Kagaku Ryoho, 13, 798805.[Medline]
-
Porsch Hallstrom,I., Svensson,D. and Blanck,A. (1991) Sex-differentiated deoxycholic acid promotion of rat liver carcinogenesis is under pituitary control. Carcinogenesis, 12, 20352040.[Abstract]
-
Qiao,D., Chen,W., Stratagoules,E.D. and Martinez,J.D. (2000) Bile acid-induced activation of activator protein-1 requires both extracellular signal-regulated kinase and protein kinase C signaling. J. Biol. Chem., 275, 1509015098.[Abstract/Free Full Text]
-
Payne,C.M., Crowley,C., Washo-stultz,D., Briehl,M., Bernstein,H., Bernstein,C., Beard,S., Holubec,H. and Warneke,J. (1998) The stress-response proteins poly(ADP-ribose) polymerase and NF-
B protect against bile salt-induced apoptosis. Cell Death Differ., 5, 623636.[ISI][Medline]
-
Morgan,W.A., Sharma,P., Kaler,B. and Bach,P.H. (1997) The modulation of protein kinase C by bile salts. Biochem. Soc. Trans., 25, 75S.[Medline]
-
Blobe,G.C., Obeid,L.M. and Hannun,Y.A. (1994) Regulation of protein kinase C and role in cancer biology. Cancer Metastasis Rev., 13, 411431.[ISI][Medline]
-
Widmann,C., Gibson,S., Jarpe,M.B. and Johnson,G.L. (1999) Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol. Rev., 79, 143180.[Abstract/Free Full Text]
-
Prives,C. and Hall,P.A. (1999) The p53 pathway. J. Pathol., 187, 112126.[ISI][Medline]
-
el-Deiry,W.S., Tokino,T., Velculescu,V.E., Levy,D.B., Parsons,R., Trent,J.M., Lin,D., Mercer,W.E., Kinzler,K.W. and Vogelstein,B. (1993) WAF1, a potential mediator of p53 tumor suppression. Cell, 75, 817825.[ISI][Medline]
-
Tso,J.Y., Sun,X.H., Kao,T.H., Reece,K.S. and Wu,R. (1985) Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res., 13, 24852502.[Abstract]
-
Lakin,N.D. and Jackson,S.P. (1999) Regulation of p53 in response to DNA damage. Oncogene, 18, 76447655.[ISI][Medline]
-
Chan,T.A., Hermeking,H., Lengauer,C., Kinzler,K.W. and Vogelstein,B. (1999) 14-3-3Sigma is required to prevent mitotic catastrophe after DNA damage [see comments]. Nature, 401, 616620.[ISI][Medline]
-
Oren,M. (1999) Regulation of the p53 tumor suppressor protein. J. Biol. Chem., 274, 3603136034.[Free Full Text]
-
Ashcroft,M. and Vousden,K.H. (1999) Regulation of p53 stability. Oncogene, 18, 76377643.[ISI][Medline]
-
Dick,L.R., Cruikshank,A.A., Grenier,L., Melandri,F.D., Nunes,S.L. and Stein,R.L. (1996) Mechanistic studies on the inactivation of the proteasome by lactacystin: a central role for clasto-lactacystin beta-lactone. J. Biol. Chem., 271, 72737276.[Abstract/Free Full Text]
-
Kudo,N., Wolff,B., Sekimoto,T., Schreiner,E.P., Yoneda,Y., Yanagida,M., Horinouchi,S. and Yoshida,M. (1998) Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1. Exp. Cell Res., 242, 540547.[ISI][Medline]
-
Murray,N.R., Davidson,L.A., Chapkin,R.S., Clay Gustafson,W., Schattenberg,D.G. and Fields,A.P. (1999) Overexpression of protein kinase C betaII induces colonic hyperproliferation and increased sensitivity to colon carcinogenesis. J. Cell Biol., 145, 699711.[Abstract/Free Full Text]
-
Perletti,G.P., Folini,M., Lin,H.C., Mischak,H., Piccinini,F. and Tashjian,A. (1996) Overexpression of protein kinase C epsilon is oncogenic in rat colonic epithelial cells. Oncogene, 12, 847854.[ISI][Medline]
-
Qiao,D., Stratagouleas,E.D. and Martinez,J.D. (2001) Activation and role of mitogen-activated protein kinases in deoxycholic acid-induced apoptosis. Carcinogenesis, 22, 3541.[Abstract/Free Full Text]
-
Meek,D.W. (1999) Mechanisms of switching on p53: a role for covalent modification? Oncogene, 18, 76667675.[ISI][Medline]
-
Song,X., Sheppard,H.M., Norman,A.W. and Liu,X. (1999) Mitogen-activated protein kinase is involved in the degradation of p53 protein in the bryostatin-1-induced differentiation of the acute promyelocytic leukemia NB4 cell line. J. Biol. Chem., 274, 16771682.[Abstract/Free Full Text]
-
Mazzoni,I.E., Said,F.A., Aloyz,R., Miller,F.D. and Kaplan,D. (1999) Ras regulates sympathetic neuron survival by suppressing the p53-mediated cell death pathway. J. Neurosci., 19, 97169727.[Abstract/Free Full Text]
-
Chernov,M.V., Ramana,C.V., Adler,V.V. and Stark,G.R. (1998) Stabilization and activation of p53 are regulated independently by different phosphorylation events. Proc. Natl Acad. Sci. USA, 95, 22842289.[Abstract/Free Full Text]
-
Gekeler,V., Boer,R., Uberall,F., Ise,W., Schubert,C., Utz,I., Hofmann,J., Sanders,K.H., Schachtele,C., Klemm,K. and Grunicke,H. (1996) Effects of the selective bisindolylmaleimide protein kinase C inhibitor GF 109203X on P-glycoprotein-mediated multidrug resistance. Br. J. Cancer, 74, 897905.[ISI][Medline]
-
Dudley,D.T., Pang,L., Decker,S.J., Bridges,A.J. and Saltiel,A.R. (1995) A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl Acad. Sci. USA, 92, 76867689.[Abstract]
-
Kirch,H.C., Flaswinkel,S., Rumpf,H., Brockmann,D. and Esche,H. (1999) Expression of human p53 requires synergistic activation of transcription from the p53 promoter by AP-1, NF-kappaB and Myc/Max. Oncogene, 18, 27282738.[ISI][Medline]
-
Colman,M.S., Afshari,C.A. and Barrett,J.C. (2000) Regulation of p53 stability and activity in response to genotoxic stress. Mutat. Res., 462, 179188.[ISI][Medline]
-
Tomlinson,I., Ilyas,M. and Novelli,M. (1997) Molecular genetics of colon cancer. Cancer Metastasis Rev., 16, 6779.[ISI][Medline]
-
Choisy-Rossi,C., Reisdorf,P. and Yonish-Rouach,E. (1999) The p53 tumor suppressor gene: structure, function and mechanism of action. Results Probl. Cell Differ., 23, 145172.[Medline]
-
Pan,Z., Damron,D., Nieminen,A.L., Bhat,M.B. and Ma,J. (2000) Depletion of intracellular Ca2+ by caffeine and ryanodine induces apoptosis of chinese hamster ovary cells transfected with ryanodine receptor. J. Biol. Chem., 275, 1997819984.[Abstract/Free Full Text]
-
Ku,W., Cheng,A. and Wang,T. (1997) Inhibition of telomerase activity by PKC inhibitors in human nasopharyngeal cancer cells in culture. Biochem. Biophys. Res. Commun., 241, 730736.[ISI][Medline]
-
Persons,D., Yazlovitskaya,E. and Pelling,J. (2000) Effect of extracellular signal-regulated kinase on p53 accumulation in response to cisplatin. J. Biol. Chem., 275, 3577835785.[Abstract/Free Full Text]
-
Canman,C.E., Lim,D.S., Cimprich,K.A., Taya,Y., Tamai,K., Sakaguchi,K., Appella,E., Kastan,M.B. and Siliciano,J.D. (1998) Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science, 281, 16771679.[Abstract/Free Full Text]
Received November 29, 2000;
revised February 7, 2001;
accepted February 22, 2001.