Departments of 1 Surgery and 3 Pathology, University of Maryland School of Medicine and 2 Baltimore Veterans Affairs Medical Center, Baltimore, Maryland 21201
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ABSTRACT |
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The p53 nuclear
phosphoprotein plays a critical role in transcriptional regulation of
target genes involved in growth arrest and apoptosis. The
natural polyamines, including spermidine, spermine, and their precursor
putrescine, are required for cell proliferation, and decreasing
cellular polyamines inhibits growth of the small intestinal mucosa. In
the current study, we investigated the mechanisms of regulation of p53
gene expression by cellular polyamines and further determined the role
of the gene product in the process of growth inhibition after polyamine
depletion. Studies were conducted both in vivo and in vitro using rats
and the IEC-6 cell line, derived from rat small intestinal crypt cells.
Levels for p53 mRNA and protein, transcription and posttranscription of
the p53 gene, and cell growth were examined. Depletion of cellular
polyamines by treatment with -difluoromethylornithine (DFMO)
increased p53 gene expression and caused growth inhibition in the
intact small intestinal mucosa and the cultured cells. Polyamine
depletion dramatically increased the stability of p53 mRNA as measured
by the mRNA half-life but had no effect on p53 gene transcription in
IEC-6 cells. Induction of p53 mRNA levels in DFMO-treated cells was
paralleled by an increase in the rate of newly synthesized p53 protein.
The stability of p53 protein was also increased after polyamine
depletion, which was associated with a decrease in Mdm2 expression.
When polyamine-deficient cells were exposed to exogenous spermidine, a
decrease in p53 gene expression preceded an increase in cellular DNA
synthesis. Inhibition of the p53 gene expression by using p53 antisense
oligodeoxyribonucleotides significantly promoted cell growth in the
presence of DFMO. These findings indicate that polyamines downregulate
p53 gene expression posttranscriptionally and that growth inhibition of
small intestinal mucosa after polyamine depletion is mediated, at least
partially, through the activation of p53 gene.
ornithine decarboxylase; growth arrest; apoptosis; messenger ribonucleic acid; protein stability; rats
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INTRODUCTION |
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REGULATION of
mammalian intestinal mucosal growth is a complex process that is
controlled and modulated by numerous factors. Epithelial renewal
originates from a small number of pluripotent stem cells, or a single
master stem cell with a cohort of daughter cells, all located in the
lower position of crypts (25, 39, 45). The stem cells
continuously divide to either renew themselves or to become committed
crypt cells that undergo a limited number of rapid divisions before
exiting the cell cycle and differentiating. The normal structure and
function of the mucosa depends on the balance between cell renewal,
growth arrest, and cell death including apoptosis (17,
19, 24). A series of observations from our previous studies
(32, 52, 53, 55) and others (26, 27) has
demonstrated that normal intestinal mucosal growth absolutely requires
the natural polyamines, including spermidine and spermine and their
precursor putrescine. Increasing cellular polyamines, either
synthesized endogenously or supplied luminally, stimulates intestinal
mucosal growth, and decreasing polyamines, by inhibiting the activity
of ornithine decarboxylase (ODC, the rate-limiting enzyme in polyamine
biosynthesis) with -difluoromethylornithine (DFMO), suppresses
mucosal growth in vivo (26, 27, 53, 55) as well as in
vitro (32, 34, 56). However, the exact mechanism by which
polyamines regulate cell growth at the molecular level is still poorly understood.
The p53 gene is responsible for control of the cell cycle, apoptosis, and the onset of cellular senescence (7, 13, 51) and may play a role in the regulation of normal intestinal mucosal growth. The product of the p53 gene is a transcription factor with a sequence-specific DNA-binding domain in the central region and a transcriptional activation domain at the NH2 terminus (20). In response to cellular stresses, the cellular levels of p53 protein are greatly increased, and the ability of p53 to bind specific DNA sequences is significantly activated (7, 13). Since expression of the p53 gene is rapid and transient, it acts as a mediator, linking short-term stress signals to growth arrest or apoptosis by regulating the activation of specific genes (7, 13, 20, 51). Several genes, including WAF1, Mdm2, GADD45, cyclin G, and PERP, have been identified as direct transcriptional targets regulated by p53 (1, 20). The p53 protein also represses various promoters lacking p53-binding sites by interactions with the basal transcription machinery, the corepressor mSin3A and histone deacetylases, or other unidentified factors (20, 31, 41). p53 is an ephemeral protein, and its half-life is approximately 10-15 min in a variety of cell types (11). In addition to transcriptional regulation, expression of the p53 gene is primarily regulated at the posttranscriptional level. Increasing evidence suggests that phosphorylation plays a major role in the regulation of both the stability of p53 protein and its DNA-binding activity (29). It has been shown that phosphorylation of human p53 at Ser-15 and Ser-20 induces conformational changes in the NH2 terminus that disrupt Mdm2 binding and lead to its stabilization (50).
We (23) have recently demonstrated that depletion of cellular polyamines by inhibiting the activity of ODC with DFMO results in the accumulation of p53 protein in small intestinal epithelial cells (IEC-6 line), which is associated with an increase in the G1 phase growth arrest but without apoptosis. The current studies were designed to address several questions regarding the regulation of p53 gene expression by cellular polyamines and the function of p53 in negative control of intestinal mucosal growth. First, we confirmed that our previous findings from cultured IEC-6 cells in vitro were also observed in intact small intestinal mucosa in vivo. Second, we characterized the process through which the cellular levels of p53 protein were increased after polyamine depletion in IEC-6 cells. Third, we determined whether increased p53 protein in polyamine-deficient cells plays a role in the inhibition of intestinal epithelial cell proliferation. Some of these data have been published in abstract form (54).
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MATERIALS AND METHODS |
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Studies in Whole Animals
Male Sprague-Dawley rats weighing between 125 and 150 g were housed in wire-bottomed, raised cages and were given water and standard laboratory rat food ad libitum. All animals were obtained from Harlan Sprague Dawley (Indianapolis, IN). Animal quarters were maintained at a temperature of 22 ± 1°C with a 12:12-h light-dark cycle. Animals were deprived of food but allowed free access to tap water for 22 h before the experiment, unless otherwise specified. Each study was carried out using five to six rats per group.The general protocol of the experiments and the methods were similar to
those described previously (55). Briefly, fed animals first were injected intraperitoneally with DFMO at the dose of 50 mg/100 g body wt, followed by 2% DFMO (a gift of Merrell Dow Research
Institute, Cincinnati, OH) in their drinking water. DFMO was then
present in the drinking water throughout the period of experiments.
Equal amounts of spermidine and spermine (Sigma, St. Louis, MO) were
suspended in 0.9% normal saline immediately before use and were given
intragastrically each in the dose of 3 mg · 100 g body
wt1 · day
1. The dose of polyamines
was divided equally into two portions, which were administered in a
volume of 0.5 ml/100 g body wt, once at 9:30 AM and again at 5:30 PM.
Control animals received the vehicle alone.
Rats were killed at 6 days of treatment by anesthesia with excess methoxyflurane. A midline abdominal incision was made to expose the small intestine, and a 4-cm intestinal segment taken 5 cm distal to the ligament of Trietz was removed, cut open, and rinsed in ice-cold saline. The mucosa was scraped from the underlying smooth muscle with a glass microscope slide, weighed, and divided into two portions. One portion was assayed to determine ODC activity, DNA synthesis, and DNA content; the other was used for measurements of p53 mRNA and protein expression.
Studies Using IEC-6 Cells
Chemicals and supplies.
Disposable cultureware was purchased from Corning Glass Works (Corning,
NY). Tissue culture media and dialyzed fetal bovine serum (dFBS) were
purchased from GIBCO (Grand Island, NY), and biochemicals were from
Sigma. The DNA probes used in these experiments included pCRII
containing a mouse p53 gene cDNA (Invitrogen, San Diego, CA) and pHcGAP
containing human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA
[American Type Culture Collection (ATCC) no. 57090]. The p53 probe
from the mouse is well characterized and has been used routinely in
studies of p53 gene expression in the rat, having >75% homology
between the species. The antibody against rat p53 protein was purchased
from Santa Cruz Biotechnology (Santa Cruz, CA). The
[-32P]dCTP (3,000 Ci/mmol) was purchased from Amersham
(Arlington Heights, IL).
Cell culture and general experimental protocol. The IEC-6 cell line was purchased from ATCC at passage 13. The cell line was derived from normal rat intestine and was developed and characterized by Quaroni et al. (40). IEC-6 cells originated from intestinal crypt cells as judged by morphological and immunologic criteria. They are nontumorigenic and retain the undifferentiated character of intestinal crypt cells.
Stock cells were maintained in T-150 flasks in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% heat-inactivated FBS, 10 µg/ml insulin, and 50 µg gentamicin sulfate/ml. Flasks were incubated at 37°C in a humidified atmosphere of 90% air-10% CO2. Stock cells were subcultured once a week at 1:20; medium was changed three times weekly. The cells were restarted from original frozen stock every seven passages. Tests for mycoplasma were routinely negative. Passages 15-20 were used in the experiments. There were no significant changes of biological function and characterization from passages 15-20. In the first series of studies, we examined the effect of cellular polyamines on the p53 gene transcription and the stability of p53 mRNA in IEC-6 cells. Cells were plated at 4 × 104 cells/cm2 and grown in control cultures or cultures containing either 5 mM DFMO alone or DFMO plus 5 µM spermidine for 6 days. The dishes were placed on ice, the monolayers were washed three times with ice-cold Dulbecco's phosphate-buffered saline (D-PBS), and then nuclei or total RNA was isolated. The rate of p53 gene transcription was measured by nuclear run-on transcription analysis, and the stability of cytoplasmic p53 mRNA was examined by determination of the mRNA half-life. Actinomycin D (5 µg/ml) was added to cultures to completely inhibit RNA synthesis, and the levels of p53 mRNA were assayed at different times after administration of actinomycin D. In the second series of studies, we examined the effect of cellular polyamines on p53 protein synthesis and its stability in IEC-6 cells. The rate of newly synthesized p53 protein was measured by using [35S]methionine-labeling technique, and the p53 stability was examined by determination of the protein half-life. Cycloheximide (50 µg/ml) was added to cultures, and p53 protein levels were assayed at different times after treatment with cycloheximide by Western blot analysis. In the third series of studies, the function of increased p53 protein in the growth inhibition was investigated in polyamine-deficient cells. Cells were initially treated with 5 mM DFMO for 4 days and then exposed to different concentrations of phosphothionate-modified p53 antisense or sense oligodeoxyribonucleotides (BIOGNOSTIK, Gottingen, Germany) for 48 h. DNA synthesis was examined by a [3H]thymidine incorporation technique.Assay for ODC activity. ODC activity in the intestinal mucosa was determined by radiometric technique in which the amount of 14CO2 liberated from DL-[L-14C]ornithine (Sigma) was estimated (42). Sample collection and the procedure of the assay were carried out according to those described in our previous publications (53, 55). Enzymatic activity was expressed as picomoles of CO2 per milligram of protein per hour.
Measurement of DNA synthesis. The rate of mucosal DNA synthesis was measured by the methods of Johnson and Guthrie (18), which included incubating the mucosal tissue in tissue culture medium containing HEPES buffer and 2 µCi/ml of [3H]thymidine (Amersham) at 37°C in an atmosphere of 95% O2-5% CO2 in a shaker-water bath. The general procedure of the method was similar to those described previously (55). The DNA synthesis in cultured cells was measured with the use of [3H]thymidine incorporation techniques, as previously described (49). Cells in 24-well plates were pulsed with 1 µCi/ml tritiated thymidine for 4 h before harvest. Cells were washed twice with cold D-PBS solution and then incubated in cold 10% TCA for 30 min at 4°C. After rinsing twice with 10% TCA, cells were dissolved in 0.5 ml of 0.5 N NaOH at 37°C in humidified air for 90 min. The incorporation of [3H]thymidine into DNA was determined by counting the aliquot of cell lysate in a Beckman liquid scintillation counter. The protein content of an aliquot of cell lysate was determined by the method as described by Bradford (3). DNA synthesis was expressed as disintegrations per minute per microgram of protein.
RNA isolation and Northern blot analysis.
Total RNA was extracted with guanidinium isothiocyanate solution and
purified by CsCl density gradient ultracentrifugation as described by
Chirgwin et al. (4). To isolate RNA from the tissue, the
mucosal scrapings were placed into 10 vol extraction buffer and
processed immediately with a Potter-Elvehjem homogenizer. Tissue
homogenate was centrifuged at 1,500 rpm for 10 min to remove tissue debris, and the supernatant was used for the isolation of total
RNA. To extract RNA from cultured cells, we washed the monolayer of
cells with D-PBS and lysed the cells in 4 M guanidinium isothiocyanate. The supernatant of the tissue or cell lysates was
brought to 2.4 M CsCl concentration and centrifuged through a 5.7 M
CsCl cushion at 150,000 g at 20°C for 24 h. After
centrifugation, the supernatant was aspirated, and the tube was cut
~0.5 cm from the bottom with a flamed scalpel. The resulting RNA
pellet was dissolved in Tris · HCl (pH 7.5) containing 1 mM
EDTA, 5% sodium laurylsarcosine, and 5% phenol (added just before
use). The purified RNA was precipitated from the aqueous phase by the
addition of 0.1 vol of 3 M sodium acetate and 2.5 vol of ethanol in
sequence. Final RNA was dissolved in water and estimated from its
ultraviolet (UV) absorbance at 260 nm using a conversion factor of 40 units. In most cases, 30 µg total cellular RNA was denatured and
fractionated electrophoretically, using a 1.2% agarose gel containing
3% formaldehyde, and transferred by blotting to nitrocellulose
filters. Blots were prehybridized for 24 h at 42°C with 5×
Denhardt's solution and 5× standard saline and sperm DNA.
cDNA probes for p53 and GAPDH were labeled with
[-32P]dCTP using a standard nick translation
procedure. Hybridization was carried out overnight at 42°C in the
same solution containing 10% dextran sulfate and
32P-labeled DNA probes. Blots were washed with two changes
of 1× standard saline citrate (SSC)/0.1% SDS at room temperature.
After the final wash, the filters were autoradiographed with
intensifying screens at
70°C. The signals were quantitated by
densitometry analysis of the autoradiograms.
Nuclear run-on transcription assays.
Nuclei were prepared according to established methods of DeBustros et
al. (5). Briefly, IEC-6 cells were suspended in
buffer A (20 mM Tris · HCl, pH 7.4, 10 mM NaCl, and
3 mM MgCl2). Nonidet P-40 was added at a final
concentration of 0.1%, and the suspension was homogenized in a sterile
Dounce homogenizer. Nuclei were pelleted by centrifugation and washed
once in buffer A. The nuclear pellet was resuspended in 40%
glycerol, 50 mM Tris · HCl, pH 8.3, 5 mM MgCl2, and
0.1 mM EDTA at 1 µg of DNA/ml and frozen at 85°C until analysis.
Nuclei isolated as described were reproducibly intact and free of
cellular debris as assessed by phase-contrast microscopy. Nuclear
transcription activity was determined by measurement of [
-32P]UTP incorporation in RNA transcripts elongated
in vitro as described by McNight and Palmiter (28).
Nuclear transcription assays were carried out in a transcription buffer
composed of 35% glycerol, 10 mM Tris · HCl, pH 7.5, 5 mM
MgCl2, 80 mM KCl, 0.1 mM EDTA, 0.5 mM dithiothreitol, 4 mM
each of ATP, GTP, and CTP, and 200 µCi of
[
-32P]UTP (800 Ci/mmol; Amersham) at 26°C for 10 min. The RNA was extracted by a modification of the guanidinium method
(4) as described in RNA isolation and Northern blot
analysis.
Western blot analysis of p53 protein. Tissue and cell samples, placed in SDS sample buffer, were homogenized, sonicated, and then centrifuged (2,000 rpm) at 4°C for 30 min. The supernatant from tissue or cell samples was boiled for 10 min and then subjected to electrophoresis on 7.5% acrylamide gels according to Laemmli (22). A 200-µg protein isolated from mucosal tissue samples or 20-µg protein from cell samples was loaded per lane equivalents. After the transfer of protein onto nitrocellulose filters, the filters were incubated for 1 h in 5% nonfat dry milk in 1× phosphate-buffered saline/Tween 20 [PBS-T: 15 mM NaH2PO4, 80 mM Na2HPO4, 1.5 M NaCl, pH 7.5, and 0.5% (vol/vol) Tween 20]. Immunologic evaluation was then performed for 1 h in 1% BSA/PBS-T buffer containing 1 µg/ml monoclonal antibody against p53 protein. The filters were subsequently washed with 1× PBS-T and incubated for 1 h with goat anti-mouse IgG antibody conjugated to peroxidase. After extensive washing with 1× PBS-T, the immunocomplexes on the filters were reacted for 1 min with chemiluminescence reagent (Du Pont NEN, NEL-100). Finally, the filters were placed in a plastic sheet protector and exposed to autoradiography film for 30 or 60 s.
Measurement of p53 protein synthesis. The p53 protein synthesis was examined by using [35S]methionine-labeling technique (2, 11). After cells were grown in control cultures and cultures containing 5 mM DFMO alone or DFMO plus spermidine for 6 days, they were washed with the methionine-free medium and incubated with the medium containing [35S]methionine (100 µCi/ml) for 2 h. The cells were rinsed with cold D-PBS containing 2 mM methionine and harvested by scraping. Cell samples were disrupted by passing through a 21-gauge syringe needle, and the suspension was centrifuged at 4°C for 10 min. The supernatant (cell lysate) was collected and incubated with a control mouse IgG together with the IgG1 protein G PLUS-agarose for 30 min on a rocker platform with a rotating device at 4°C. Beads were isolated by centrifugation, and the preclear cell lysate was transferred into a new tube. The cell lysate (400 µg) was incubated with anti-p53 antibody (4 µg) for 2 h at 4°C. The protein G PLUS-agarose was added, and the samples were incubated overnight. Immunoprecipitates were carefully collected after centrifugation at 2,500 rpm for 5 min, and pellets were washed with cold PBS and resuspended in 30 µl of 1× electrophoresis sample buffer. The supernatant was analyzed by SDS-PAGE followed by autoradiography.
Immunohistochemical staining. Immunohistochemical staining for p53 protein was performed in IEC-6 cells by the indirect immunoperoxidase method as described previously (23). Cells were plated on 22 × 22-mm glass coverslips, which were placed in 35-mm dishes. After different treatments, cells were washed with D-PBS and then with D-PBS without Ca2+ and Mg2+ and fixed for 5 min at room temperature in 4% paraformaldehyde diluted with D-PBS. The cells were postfixed for 5 min with ice-cold methanol. The cells were rehydrated in D-PBS without Ca2+ and Mg2+ for 30 min at room temperature and were then incubated with the primary antibody against p53 protein at a dilution of 1:50 in humidified chambers for 24 h at 4°C. Nonspecific slides were incubated without antibody to p53 protein. The bound antibody was visualized with biotinylated second antibody and avidin-biotin complexes (ABC). The slides were counterstained with hematoxylin, mounted, and reviewed with an Olympus microscope.
Statistics. All data are expressed as means ± SE from six dishes. Autoradiographic and immunohistochemical staining results were repeated three times. The significance of the difference between means was determined by analysis of variance. The level of significance was determined using Duncan's multiple range test (14).
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RESULTS |
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Effect of Polyamine Depletion on p53 Gene Expression in Intact Small Intestinal Mucosa In Vivo
Inhibition of polyamine synthesis by treatment with DFMO significantly suppressed small intestinal mucosal growth in rats (Fig. 1A). The decrease in the rate of [3H]thymidine incorporation was observed on day 4 (data not shown), and the maximal inhibition occurred on day 6 (Fig. 1Ab) after beginning treatment with DFMO. Decreased DNA synthesis was paralleled by the decrease in total mucosal DNA content (Fig. 1Ac). Oral administration of spermidine and spermine (each at 3 mg · 100 g body wt
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Inhibition of polyamine synthesis also increased expression of the p53 gene in small intestinal mucosa (Fig. 1B). Basal levels of p53 mRNA and protein in the mucosa were low but significantly increased by treatment with DFMO. The concentrations of p53 mRNA and protein were greater than two times control values at 6 days after administration of DFMO. Polyamines (spermidine and spermine) given together with DFMO prevented increased expression of the p53 gene. The mucosal levels of p53 mRNA and protein in rats treated with DFMO plus polyamines were indistinguishable from those in control rats. These data indicate that growth inhibition in intact small intestinal mucosa after polyamine depletion is associated with an increase in p53 gene expression in vivo.
Effect of Polyamine Depletion on p53 Gene Transcription and p53 mRNA Stability in IEC-6 Cells
The IEC-6 cell line represents rat small intestinal crypt cells and provides an appropriate model to investigate intestinal mucosal epithelial cell proliferation in vitro. Inhibition of ODC activity by treatment with 5 mM DFMO almost completely depleted cellular polyamine in IEC-6 cells. The levels of putrescine and spermidine were undetectable at 4, 6, and 12 days after treatment with DFMO, and spermine was decreased by ~60%. Inhibition of polyamine synthesis by DFMO also resulted in the G1 phase growth arrest in IEC-6 cells (data not shown). Similar results have been published previously (23, 56).As shown in Fig. 2A, polyamine
depletion by treatment with DFMO significantly increased the levels for
p53 mRNA, which was completely prevented by spermidine given together
with DFMO. To test the possibility that the increase in p53 mRNA level
in polyamine-deficient cells results from an increase in the mRNA
synthesis, changes in the rate of p53 gene transcription were examined
by using nuclear run-on transcription assays. Inhibition of polyamine
synthesis by DFMO did not increase p53 gene transcription in IEC-6
cells (Fig. 2B). There were no significant differences in
the rate of p53 gene transcription between control cells and cells
exposed to DFMO in the presence or absence of spermidine for 6 days. We also measured the rate of p53 gene transcription in cells exposed to
DFMO for 2 and 4 days and demonstrated that the results were similar to
those observed after 6-day treatment (data not shown). These results
clearly indicate that cellular polyamines play little role in the
regulation of p53 gene transcription and that the increase in
steady-state levels of p53 mRNA after polyamine depletion is related to
a mechanism other than the stimulation of p53 gene transcription in
intestinal epithelial cells.
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To test the involvement of p53 mRNA stability in this process, we
determined the effect of polyamine depletion on p53 mRNA half-life in
IEC-6 cells. As can be seen in Fig. 3,
depletion of cellular polyamines by DFMO significantly increased the
stability of p53 mRNA. In control cells, p53 mRNA levels declined
rapidly after inhibition of gene transcription by addition of
actinomycin D (Fig. 3Aa). The half-life of p53 mRNA in
control cells was ~45 min. However, the stability of p53 mRNA was
dramatically increased by polyamine depletion with a half-life of >18
h (Fig. 3, Ab and B). Increased half-life of p53
mRNA was prevented when spermidine was given together with DFMO (Fig.
3, Ac and B). The half-life of p53 mRNA in cells
treated with DFMO plus spermidine was ~48 min, similar to that of
controls (without DFMO). These findings suggest that polyamines
regulate the p53 gene expression posttranscriptionally in intestinal
epithelial cells and that depletion of cellular polyamines induces p53
mRNA levels primarily through the increase in its stability.
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Changes in p53 Protein Synthesis and Stability After Polyamine Depletion
Increased p53 mRNA in DFMO-treated cells was paralleled by an increase in p53 protein synthesis (Fig. 4A). The rate of newly synthesized p53 protein was increased by ~80% in cells exposed to DFMO for 6 days. Similar changes in p53 protein synthesis were also observed in cells exposed to DFMO for 4 days (data not shown). The p53 protein synthesis was returned to normal levels when DFMO was given together with spermidine. Consistent with the augmenting effect on p53 protein synthesis, p53 protein levels increased significantly in DFMO-treated cells, which was prevented by exogenous spermidine (Fig. 4B).
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Results presented in Fig. 5 showed that
polyamine depletion also increased the stability of p53 protein in
intestinal epithelial cells. The levels of p53 protein in control cells
declined rapidly after inhibition of protein synthesis by
administration of cycloheximide. The half-life of p53 protein in
control IEC-6 cells was ~15 min (Fig. 5, Aa and
B) and increased to ~38 min in cells exposed to DFMO for 6 days (Fig. 5, Ab and B). When DFMO was given
together with spermidine, p53 protein levels decreased at the rate
similar to that observed in controls, with a half-life of ~18 min.
These data indicate that cellular polyamines are essential for the
degradation of p53 protein in IEC-6 cells and that induced accumulation
of p53 protein in polyamine-deficient cells results, at least
partially, from the protein stabilization.
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Effect of Polyamine Depletion on Mdm2 Expression
To determine the involvement of Mdm2 in the process of increased stability of p53 protein after polyamine depletion, expression of Mdm2 protein was examined in cells grown in the presence or absence of DFMO. As can be seen in Fig. 6, depletion of cellular polyamines by DFMO significantly decreased the content of Mdm2 protein in IEC-6 cells. The levels of Mdm2 protein were decreased by ~60% on days 4, 6, and 12 after exposure to DFMO. The Mdm2 protein content returned to normal levels when DFMO was given together with spermidine. These results suggest that increased stability of p53 protein after depleted cellular polyamines is mediated through a process involving Mdm2.
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Effect of Disruption of p53 Expression on Growth in Polyamine-Deficient Cells
To elucidate whether increased accumulation of p53 protein plays a role in the process of growth inhibition after polyamine depletion, two types of experiments were carried out in DFMO-treated cells. In the first study, we examined the time course of p53 gene expression and cell growth when polyamine-deficient cells are exposed to exogenous spermidine. Cells were initially treated with 5 mM DFMO for 4 days and then exposed to 5 µM spermidine. The changes in both p53 gene expression and cellular DNA synthesis were measured at various times after spermidine was added to the medium containing DFMO. Depletion of cellular polyamines by administration of DFMO for 4 days increased the steady-state levels of p53 mRNA (Fig. 7Aa) and protein (Fig. 7Ab). When these polyamine-deficient cells were exposed to exogenous spermidine, prevention of increased p53 mRNA and protein preceded the beginning of cellular DNA synthesis as measured by [3H]thymidine incorporation. The significant decreases in p53 mRNA and protein levels were observed at 12 h after exposure to exogenous spermidine (Fig. 7A), whereas the increased rate of DNA synthesis occurred at 24 h after administration of spermidine (Fig. 7B). These findings indicate that the restoration of cell growth by exogenous spermidine in polyamine-deficient cells is preceded by a decrease in p53 gene expression.
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In the second study, effect of inhibition of p53 expression by using
p53 antisense oligodeoxyribonucleotides on cell growth was examined in
polyamine-deficient cells. Cells were grown in the presence of 5 mM
DFMO for 4 days, and DNA synthesis was assayed by
[3H]thymidine incorporation 48 h after
administration of different concentrations (0.5-2 µM) of p53
antisense or sense oligomers. Exposure of polyamine-deficient cells to
p53 antisense oligomers partially but significantly increased DNA
synthesis (Fig.
8A) in the
presence of DFMO. Treatment with p53 antisense oligomers also inhibited
p53 protein expression in DFMO-treated cells (Fig. 8, B and
C). When DFMO-treated cells were exposed to 2 µM antisense oligomers for 48 h, increased p53 protein levels were completely prevented as measured by Western blot analysis (Fig.
8B) and immunohistochemical staining (Fig.
8C). Treatment with sense p53 oligomers at the same concentrations, however, showed no significant effects on DNA
synthesis (Fig. 8A) and p53 protein expression (Fig. 8,
B and C). These results provide direct evidence
that increased expression of the p53 gene plays a critical role in
growth inhibition of intestinal epithelial cells after polyamine
depletion.
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DISCUSSION |
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The maintenance of intestinal mucosal epithelial integrity requires cellular polyamines that regulate expression of the genes involved in proliferation, growth arrest, differentiation, and apoptosis (26, 27, 36, 38). The present studies advance our previous observations (23) that inhibition of polyamine synthesis results in the accumulation of p53 in cultured IEC cells by demonstrating that DFMO-induced polyamine depletion increases levels of p53 mRNA and protein in intact small intestinal mucosa in vivo (Fig. 1). This increase in expression of the p53 gene after polyamine depletion was associated with an inhibition of mucosal growth. The most significant of the new findings reported in this study, however, is that polyamine depletion induces expression of the p53 gene through a posttranscriptional mechanism in intestinal epithelial cells. Depletion of cellular polyamines by DFMO had no effect on the rate of p53 gene transcription (Fig. 2) but dramatically increased the half-life of p53 mRNA (Fig. 3), leading to the elevation of the steady-state levels of p53 mRNA and the induction of newly synthesized p53 protein in IEC-6 cells (Fig. 4). On the other hand, polyamine depletion decreased Mdm2 expression and slowed p53 protein degradation in IEC-6 cells (Figs. 5 and 6), which also contributes to the increase in p53.
At physiological pH, putrescine, spermidine, and spermine possess two,
three, and four positive charges, respectively (47). These
compounds are believed to exert their effects by binding to negatively
charged macromolecules such as DNA, RNA, and proteins to influence the
chromatin structure and sequence-specific DNA- or RNA-protein
interactions, which alter the rate of gene transcription and the
stabilization of mRNAs and proteins (36, 38, 47). Increasing evidence indicates that cellular polyamines play different roles in the regulation of expression of various genes in a variety of
cell types, including intestinal epithelial cells (32, 33, 58). It has been shown that polyamines are essential for
c-myc and c-jun mRNA synthesis in IEC-6 cells and
that depletion of cellular polyamines decreases the transcription rates
of these two genes but has no effect on their posttranscription
(33). In contrast, inhibition of polyamine synthesis
significantly increases the stability of transforming growth factor-
(TGF-
) mRNA without affecting the rate of transcription of this gene
(32).
The findings reported here clearly indicate that polyamines negatively regulate posttranscription but not transcription of the p53 gene in IEC-6 cells. As noted in Figs. 2 and 3, administration of DFMO dramatically increased the half-life of p53 mRNA, although it did not affect the rate of transcription of the p53 gene. This prolonged half-life leads to the accumulation of p53 mRNA, which is paralleled by an increase in p53 protein synthesis. Because the increases in p53 mRNA stability and the resultant protein synthesis in DFMO-treated cells are completely prevented by the addition of exogenous spermidine, these observed changes in the posttranscriptional regulation of the p53 gene must be related to polyamine depletion rather than to the nonspecific effect of DFMO.
Posttranscriptional regulation, especially modulation of mRNA stability, has been shown to play an important role in gene expression (43). The turnover rate of a given mRNA can be determined by interactions of trans-acting factors with specific cis-element(s) located within 3'-untranslated regions (3'-UTR) (16, 43). Many labile mRNAs, including those that encode transcription factors, contain AU-rich elements (AREs) in their 3'-UTR (16, 43). The presence of a reiterated pentamer (5'-AUUUA-3') in many AREs is associated with rapid mRNA turnover and translation attenuation (10, 16, 35, 59). Deletion of the ARE region enhances mRNA stability, and insertion of the region into the 3'-UTR of a normally stable globin mRNA significantly destabilizes it (35, 46, 59). A variety of AUUUA-binding proteins have been identified in different cell types (16, 46), although the mechanisms of how these proteins affect mRNA turnover are unclear. Whether an ARE region in the p53 3'-UTR is critical for the stabilization of p53 mRNA after polyamine depletion in intestinal epithelial cells remains to be elucidated.
The data from the current studies also reveal that polyamine depletion decreased Mdm2 expression and slowed p53 protein degradation (Figs. 5 and 6). Mdm2 was originally identified as an amplified gene in a spontaneously transformed derivative of BALB/c cell line 3T3 DM (9). An attractive model has been presented in which Mdm2 is a major regulator of p53 (15). The product of Mdm2 gene directly binds to p53 and forms a complex to promote p53 protein degradation and inhibit its transcriptional activity, thereby creating an autoregulatory feedback loop that regulates p53 expression and activity (15, 30, 57). Mdm2 is a transcriptional target of p53, and the p53-responsive elements have been identified in the intronic promoter of the Mdm2 gene (15, 57). Interaction of p53 with these binding sides in the Mdm2 promoter has been shown to induce transcription of the Mdm2 gene in different cell types, through which p53 itself initiates its own destruction (37, 57). It is possible that polyamine depletion downregulates Mdm2 protein expression through a p53-independent pathway in IEC-6 cells and that decreased Mdm2 could be partially responsible for the increase in p53 protein stability.
Increased accumulation of p53 through posttranscriptional regulation plays an important role in the process of growth inhibition after polyamine depletion in intestinal epithelial cells. Although cell homeostasis is a balance between proliferation, growth arrest, and cell death including apoptosis, studies on negative growth control have not attracted considerable interest until recently. Treatment with DFMO increases expression of the p53 gene in small intestinal mucosa both in vivo and in vitro, which is completely prevented by the addition of exogenous polyamines. Polyamine depletion also induces G1 phase growth arrest and alters susceptibility to apoptotic stimuli but does not directly induce apoptosis in IEC-6 cells (23, 24). The results presented in Fig. 7 show that the decrease in p53 protein precedes the initiation of DNA synthesis as measured by [3H]thymidine incorporation when polyamine-deficient cells were exposed to exogenous spermidine. Furthermore, inhibition of p53 expression by treatment with p53 antisense oligonucleotide partially but significantly promoted cell proliferation in the presence of DFMO (Fig. 8). These findings provide direct evidence to support the hypothesis that decreasing cellular polyamines induces p53, resulting in the inhibition of normal small intestinal mucosal growth.
The cell cycle regulatory pathway responsible for the growth arrest induced by p53 in normal intestinal epithelial cells after polyamine depletion is obscure and may be related to the cyclin-dependent kinase (cdk) inhibitor p21 (12). There is little doubt that the combined effects of kinases, phosphatases, and inhibitory proteins, mediated by protein partnering and positive- and negative-acting phosphorylation, elegantly control progression through the cell cycle. One of the well-characterized systems is the cdk-pRb signaling pathway, which is activated in the G1 phase and initiates progression toward S phase (6, 44). p53 has been shown to activate transcription of the p21cip gene through interaction with two p53-responsive elements located in the p21cip promoter (48). Cells lacking p21cip expression have an impaired p53-dependent response to various stimuli (8, 12). It is possible that increased p53 induces p21, which inhibits cyclin E-cdk2 and/or cyclin D1-cdk4 activities and prevents phosphorylation of Rb protein, blocking the release of E2F and thus inhibiting the G1-S transition during the cell cycle after polyamine depletion in intestinal epithelial cells. In support of this contention, Kramer et al. (21) reported that depletion of cellular polyamines by treatment with polyamine analog, N1-N11-diethylnorspermidine, results in the G1 phase growth arrest via the p53-p21cip-Rb pathway in human melanoma cells.
In summary, we propose a model delineating the regulation of expression
of the p53 gene by cellular polyamines and the involvement of p53 in
the process of growth inhibition of normal small intestinal mucosa
after polyamine depletion (Fig. 9). In
this model, polyamines negatively regulate posttranscription of the p53
gene, and the activation of p53 expression plays a critical role in the
negative control of the small intestinal mucosal growth. Depletion of
cellular polyamines, either by inhibition of their synthesis,
stimulation of catabolism, or suppression of polyamine uptake, enhances
expression of the p53 gene via both the stabilization of p53 mRNA and
the decrease in p53 protein degradation. The resultant accumulation of
p53 in polyamine-deficient cells activates the transcription of cell
cycle arrest genes such as p21cip, which then
blocks the G1-S phase transition, decreases proliferation, and inhibits mucosal growth of the small intestine.
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ACKNOWLEDGEMENTS |
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This work was supported by Merit Review Grants from the Department of Veterans Affairs to J-Y. Wang and B. L. Bass and by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-57819 to J-Y. Wang. J-Y. Wang is a Research Career Scientist, Medical Research Service, Department of Veterans Affairs.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J.-Y. Wang, Dept. of Surgery, Baltimore Veterans Affairs Medical Center, 10 N. Greene St., Baltimore, MD 21201 (E-mail: jwang{at}smail.umaryland.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 20 March 2001; accepted in final form 3 May 2001.
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