Department of Surgery, University of Maryland School of Medicine and Baltimore Veterans Affairs Medical Center, Baltimore, Maryland 21201
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ABSTRACT |
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The nuclear phosphoprotein p53 acts as a transcription factor
and is involved in growth inhibition and apoptosis. The present study
was designed to examine the effect of decreasing cellular polyamines on
p53 gene expression and apoptosis in small intestinal epithelial
(IEC-6) cells. Cells were grown in DMEM containing 5% dialyzed fetal
bovine serum in the presence or absence of -difluoromethylornithine (DFMO), a specific inhibitor of polyamine biosynthesis, for 4, 6, and
12 days. The cellular polyamines putrescine, spermidine, and spermine
in DFMO-treated cells decreased dramatically at 4 days and remained
depleted thereafter. Polyamine depletion by DFMO was accompanied by a
significant increase in expression of the p53 gene. The p53 mRNA levels
increased 4 days after exposure to DFMO, and the maximum increases
occurred at 6 and 12 days after exposure. Increased levels of p53 mRNA
in DFMO-treated cells were paralleled by increases in p53 protein.
Polyamines given together with DFMO completely prevented increased
expression of the p53 gene. Increased expression of the p53 gene in
DFMO-treated cells was associated with a significant increase in
G1 phase growth arrest. In
contrast, no features of programmmed cell death were identified after
polyamine depletion: no internucleosomal DNA fragmentation was
observed, and no morphological features of apoptosis were evident in
cells exposed to DFMO for 4, 6, and 12 days. These results indicate
that 1) decreasing cellular
polyamines increases expression of the p53 gene and
2) activation of p53 gene expression after polyamine depletion does not induce apoptosis in intestinal crypt
cells. These findings suggest that increased expression of the p53 gene
may play an important role in growth inhibition caused by polyamine depletion.
growth inhibition; proliferation; tumor suppressor gene; ornithine decarboxylase; IEC-6 cells
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INTRODUCTION |
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THE EPITHELIAL CELLS of the gastrointestinal mucosa are
among the most rapidly proliferating cells in the body (14, 21). Normal
structure and function of the mucosa depend on a regulated rate of
division of proliferating cells in the mucous neck region in the
stomach and the crypts in the small intestine (19, 21). Inhibition of
intestinal mucosal growth occurs commonly in critical illness and leads
to diarrhea, malabsorption, delayed healing, and impaired barrier
function. Increasing evidence has indicated that the cellular
polyamines spermidine and spermine and their precursor putrescine are
necessary for normal mucosal growth and that decreasing cellular
polyamine levels inhibit cell renewal (15, 20, 21, 33). Intracellular
polyamine levels are highly regulated and primarily dependent on the
activation or inhibition of ornithine decarboxylase (ODC), which
catalyzes the first step in polyamine biosynthesis (13, 26). Our
previous studies have shown that depletion of cellular polyamines by
inhibition of ODC with -difluoromethylornithine (DFMO) significantly
decreases mucosal growth in vivo (34) as well as in vitro (36), but the
exact mechanism by which polyamine depletion results in growth inhibition remains to be demonstrated.
Cell homeostasis is regulated by a balance among proliferation, growth arrest, and apoptosis. The recognition that negative growth control must be elucidated to comprehend the mechanisms by which appropriate cell numbers are maintained has attracted considerable interest. The p53 gene encodes for a nuclear phosphoprotein, which acts as a transcription factor and has been shown to be involved in the processes of growth inhibition and apoptosis (6, 10, 18, 22). The p53 protein is present in low concentrations in normal cells and is a negative factor for cell cycle control, since progression from the G1 to the S phase is often blocked in cells expressing high levels of this protein. Induction of p53 expression by transfection with a conditional p53 expression vector, for example, inhibits cell cycle progression in a glioblastoma tumor cell line (22). When growth-arrested cells are stimulated to proliferate, induction of p53 expression inhibits progression from the G0/G1 to the S phase (18, 22).
Apoptosis is an energy-dependent and highly regulated process by which cells die without releasing their contents and without eliciting inflammation (17, 28). Apoptosis is absolutely required for the natural development and homeostasis of tissues in complex multicellular organisms (40). As such, it is likely that apoptosis is implicated in the regulation of normal growth in the gastrointestinal mucosa. It has been shown that stimulation of expression of the p53 gene induces growth arrest and/or apoptosis in a number of cell types (17, 29). The p53 protein can simultaneously induce the genetic programs of G1 phase growth arrest and apoptosis within the same cell type in which apoptosis can proceed in G1-arrested or cycling cells (17).
To our knowledge, there are no studies concerning changes in p53 gene expression and apoptosis after polyamine depletion in intestinal epithelial cells. Given that polyamine depletion abolishes intestinal epithelial cell growth, we investigate the mechanism of this growth arrest and the possible role of p53. The immediate goal of the present study was to determine whether decreasing cellular polyamine levels by treatment with DFMO induces expression of the p53 gene and apoptosis in cultured normal rat small intestinal crypt (IEC-6) cells.
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MATERIALS AND METHODS |
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Chemicals and supplies.
Disposable culture ware was purchased from Corning Glass Works
(Corning, NY). Tissue culture media and dialyzed fetal bovine serum
(FBS) were purchased from GIBCO (Grand Island, NY), and biochemicals
were obtained from Sigma Chemical (St. Louis, MO). The DNA probes used
in these experiments included
pCRTMII containing a mouse p53
gene cDNA (Invitrogen, San Diego, CA), pBR322 containing a human
genomic fragment coding for retinoblastoma susceptibility
(Rb) gene [no. 57450, American
Type Culture Collection (ATCC)], and pHcGAP containing human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (no. 57090, ATCC). The p53 probe from the mouse is well characterized and has been
used routinely in studies of p53 gene expression in the rat; homology
between the species is >75%. 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). DFMO was
the kind gift of the Merrell Dow Research Institute (Cincinnati, OH).
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. (27). IEC-6 cells originated from intestinal crypt cells as judged by morphological and immunological criteria. They are nontumorigenic and retain the undifferentiated character of epithelial stem cells.
Stock cells were maintained in T-150 flasks in DMEM supplemented with 5% heat-inactivated FBS, insulin (10 µg/ml), and gentamicin sulfate (50 µg/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 passage 15 to 20. In the first series of studies we examined whether depletion of cellular polyamines by DFMO could alter expression of the p53 gene in IEC-6 cells. The general protocol of the experiments and the methods were similar to those described previously (36). Briefly, IEC-6 cells were plated at 6.25 × 104 cells/cm2 and grown in control cultures or cultures containing 5 mM DFMO or DFMO plus spermidine (5 µM) for 4, 6, and 12 days. The dishes were placed on ice, the monolayers were washed three times with ice-cold Dulbecco's PBS (D-PBS), and then different solutions were added according to the assays to be conducted. In the second series of studies we examined whether increased expression of the p53 gene after polyamine depletion was associated with programmed cell death in IEC-6 cells. Changes in internucleosomal DNA fragmentation, the morphological features of apoptosis, and the distribution of cell cycle were measured at various times after exposure to DFMO with or without spermidine.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. (5). Briefly, the monolayer of cells was washed with
D-PBS and lysed in 4 M guanidinium isothiocyanate. The lysates were
brought to 2.4 M CsCl 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 absorbance at 260 nm by using a conversion factor
of 40 units. In most cases, 30 µg of total cellular RNA were
denatured and fractionated electrophoretically by 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 sperm
DNA. cDNA probes for p53, Rb, and
GAPDH were labeled with
[-32P]dCTP by 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× saline sodium
citrate-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.
Western blot analysis of p53 protein. Cell samples, dissolved in SDS sample buffer, were sonicated for 20 s and centrifuged at 2,000 rpm for 15 min. The supernatant was boiled for 10 min and then subjected to electrophoresis on 7.5% acrylamide gels according to Laemmli (16). Each lane was loaded with 20 µg of protein equivalents. Briefly, after the transfer of protein onto nitrocellulose filters, the filters were incubated for 1 h in 5% nonfat dry milk in 1× PBS-Tween 20 (PBS-T) containing 15 mM NaH2PO4, 80 mM Na2HPO4, and 1.5 M NaCl, pH 7.5, and 0.5% (vol/vol) Tween 20. Immunological 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 (NEL-100, Du Pont NEN). Finally, the filters were placed in a plastic sheet protector and exposed to autoradiography film for 30 or 60 s.
Immunohistochemical staining. Immunohistochemical staining for p53 protein was performed in IEC-6 cells by the indirect immunoperoxidase method, as described previously (12). Cells were plated at 6.25 × 104 cells/cm2 on 22 × 22-mm glass coverslips, which were placed in 35-mm dishes in medium consisting of DMEM, 5% dialyzed FBS, 10 µg/ml insulin, and 50 µg/ml gentamicin sulfate. DFMO (5 mM) with or without 5 µM spermidine was added as treatment. At 6 days after initial plating, the 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, rehydrated in D-PBS without Ca2+ and Mg2+ for 30 min at room temperature, and then incubated with goat polyclonal IgG raised 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 anti-goat IgG and avidin-biotin complexes. The slides were counterstained with hematoxylin, mounted, and viewed with an Olympus microscope.
Determination of internucleosomal DNA cleavage. Internucleosomal DNA fragmentation was assayed by a modification of previously described methods (4). After cells were grown in the presence of DFMO with or without spermidine for various times, they were harvested and washed twice with cold PBS at 4°C. Cells were suspended in lysis solution containing 5 mM Tris · HCl, 20 mM EDTA, and 5% (vol/vol) Triton X-100 for 20 min on ice. The remaining steps for DNA fragmentation analysis were performed exactly as described previously (1). DNA samples were analyzed by electrophoresis in a 1.5% agarose slab gel containing 0.2% µg/ml ethidium bromide and visualized under ultraviolet illumination.
Flow cytometry analysis for cell cycle distribution. Cell sample preparation and propidium iodide staining for flow cytometry analysis were performed according to the method described by Nicoletti and Cooper (23). Briefly, IEC-6 cells were cultured in 10-cm plates at 6.25 × 104 cells/cm2 and treated with DFMO with or without spermidine. Cells were harvested by trypsinization, washed twice in D-PBS, and fixed in 70% ethanol diluted with D-PBS. Cells were incubated in D-PBS containing RNase (100 µg/ml) and propidium iodide (40 µg/ml) at 37°C for 1 h before flow cytometry analysis. Cell cycle distribution was determined using a Coulter Epics V instrument with an argon laser set to excite at 488 nm. The results were analyzed using Elite 4.0 software (Phoenix Flow System).
Polyamine analysis.
The nuclear polyamine content was analyzed by HPLC, as described
previously (36). After washing the monolayers three times with ice-cold
D-PBS, we added 0.5 M perchloric acid and then froze the monolayers at
80°C until ready for extraction, dansylation, and HPLC. The
standard curve encompassed 0.31-10 µM. Values that fell >25%
below the curve were considered not detectable. Protein was determined
by the Bradford method (2). The results are expressed as nanomoles of
polyamines per milligram of protein.
Statistics. Values are means ± SE from six dishes. Autoradiographic results were repeated three times. The significance of the difference between means was determined by ANOVA. The level of significance was determined using Duncan's multiple range test (11).
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RESULTS |
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Effect of polyamine depletion on expression of the p53 gene.
Administration of 5 mM DFMO, which totally inhibited ODC activity (36,
39), almost completely depleted cellular polyamines in IEC-6 cells
(Fig. 1). The levels of
putrescine and spermidine were undetectable at 4, 6, and 12 days after
DFMO treatment. Spermine was less sensitive to the inhibition of ODC
but was decreased by >60% in cells exposed to DFMO for 4, 6, and 12 days.
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Effect of serum starvation on cellular polyamines and p53 gene
expression.
To test the specificity of increased expression of the p53 gene after
polyamine depletion, we examined the effect of serum starvation on
expression of the p53 gene. Our previous study demonstrated that
starvation of IEC-6 cells by the removal of serum for 72 h decreased
DNA synthesis by >75% (data not shown). In this study it has been
shown that there was no significant decrease in cellular polyamines
when cells were grown in the absence of serum for 72 h (data not
shown). Consistent with the effect on cellular polyamines, serum-deprived quiescent IEC-6 cells were not associated with an
increased expression of the p53 gene (Fig.
6). In fact, p53 mRNA levels slightly
decreased at 48 and 72 h after serum deprivation. These results clearly
show that serum-deprived quiescent IEC-6 cells do not decrease cellular
polyamines and therefore have no effect on p53 gene expression. These
findings suggest that increased expression of the p53 gene in the
DFMO-treated cells is specifically related to polyamine depletion and
does not result simply from decreased growth.
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Changes in cell proliferation and apoptosis after polyamine
depletion.
Figure 8 shows that increased expression of
the p53 gene in the DFMO-treated IEC-6 cells was associated with a
significant decrease in cell numbers. With the activation of p53 gene
expression after polyamine depletion, cell numbers were significantly
decreased at 4 days, an effect maintained for up to 12 days. In the
presence of DFMO, increased p53 gene expression and decreased cell
numbers were completely prevented by addition of exogenous spermidine. The level of p53 protein (Figs. 3 and 5) and the number of cells were
indistinguishable in cells exposed to DFMO plus spermidine and control
cells.
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DISCUSSION |
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Cell proliferation in the intestinal mucosa is dependent on the supply of polyamines to the dividing cells in the crypts (20, 21, 30, 35). Decreasing cellular polyamines significantly inhibits cell renewal, but the mechanism involved in growth inhibition remains to be elucidated. In the present study we investigated the effect of inhibition of polyamine biosynthesis on p53 gene expression and apoptosis in intestinal epithelial cells. Our results clearly show that depletion of cellular polyamines by treatment with DFMO significantly increases p53 mRNA levels, which were paralleled by increases in p53 protein (Figs. 2 and 3). The activation of p53 gene expression after polyamine depletion is associated with a significant increase in G1 phase growth arrest but without apoptosis (Figs. 9 and 10). These findings suggest that expression of the p53 gene is highly regulated by cellular polyamines and may play an important role in the process of mucosal growth inhibition after polyamine depletion.
Although exact roles for cellular polyamines in specific biochemical events related to cell proliferation at the molecular level are largely unknown, several studies have indicated that expression of the protooncogenes c-fos, c-myc, and c-jun is at least partially involved in the early modulation of mucosal growth stimulation by polyamines (36, 37). Induction of mucosal growth in vivo (31) as well as in vitro (36) is accompanied by a significant increase in protooncogene expression after an increase in cellular polyamines, which precedes the induction of DNA synthesis. Polyamines are required for the protooncogene transcription, and depletion of cellular polyamines prevents increases in protooncogene expression and cell proliferation (25). However, polyamine-deficient cells have been shown to continuously maintain a high basal level of protooncogene expression (25, 36), indicating that growth inhibition after polyamine depletion must result from a mechanism other than a simple decrease in protooncogene expression. The change in activation of protooncogene expression is mainly relevant to the process of growth stimulation by polyamines but plays a minor role in growth inhibition after polyamine depletion (25, 36).
The recognition that negative growth control, including growth inhibition and programmed cell death, must be understood to comprehend how appropriate cell numbers are maintained in normal mucosa and how alterations in any part of the equation contributes to mucosal atrophy after polyamine depletion led us to consider the possibility that growth inhibition in DFMO-treated cells could be due in part to the activation of growth-inhibiting gene expression. The results reported here support our hypothesis and indicate that administration of DFMO not only completely depletes cellular polyamines but also significantly increases p53 gene expression in intestinal epithelial cells. Increased expression of the p53 gene in DFMO-treated cells is related to polyamine depletion rather than to a nonspecific effect of DFMO, because polyamine given together with DFMO prevents the increase in the levels of p53 mRNA and protein.
Three experiments were performed to further characterize the relationship between cellular polyamines and induced expression of the p53 gene in intestinal epithelial cells. First, we demonstrated that polyamine depletion has no effect on Rb gene expression (Fig. 2). Second, cellular polyamines are not decreased in serum-deprived quiescent IEC-6 cells and are not associated with the activation of p53 gene expression (Fig. 6). Third, we compared polyamine-deficient cells with control cells in which growth was inhibited at confluence. As can be seen in Figs. 2 and 8, although depletion of polyamines by DFMO significantly increases p53 gene expression, there are no increases in p53 mRNA and protein levels in control cells in which growth was inhibited at confluence at 6 and 12 days after initial plating. These results indicate that polyamines have a specific effect on p53 gene expression and that increased levels of p53 mRNA and protein in DFMO-treated cells result from decreasing cellular polyamines and are not a secondary effect of growth inhibition.
To elucidate the biological significance of increased p53 protein after
polyamine depletion, we examined the change in programmed cell death in
DFMO-treated cells. Data presented in Figs. 9 and 10 show that
depletion of cellular polyamines does not induce apoptosis. These
results are consistent with the results of Casero et al. (3), which
demonstrated that polyamine depletion alone did not result in
programmed cell death in the human lung tumor cell line NCI H157.
However, other studies indicate that the cellular polyamines are
involved in the process of apoptosis. Decreased cellular polyamines and
increased activity of the polyamine catabolic enzyme
spermidine/spermine
N1-acetytransferase
have been shown in dexamethasone- and polyamine analog-induced
apoptosis (7, 9). An imbalance of polyamine metabolism may be a trigger
of apoptosis in heat shock treatment- and -irradiation-induced cell
death, in which increases in ODC mRNA and activity are observed without
subsequent increases in cellular polyamine levels (8).
The nature of the molecular mechanisms that activate the expression of
the p53 gene after polyamine depletion remains to be demonstrated.
Although increased p53 protein is paralleled by a significant increase
in p53 mRNA levels in DFMO-treated cells, it is not clear whether
increased p53 mRNA is due to an increase in the gene transcription or
results from the alteration of the mRNA stabilization. There is no
doubt that cellular polyamines play different roles in the expression
of various growth-related genes and that their effects are cell type
dependent (36, 38). It has been shown that cellular polyamines are
absolutely required for c-myc and
c-jun mRNA synthesis in IEC-6 cells
and that depletion of cellular polyamines by treatment with DFMO
significantly decreases the transcription rates of these two genes but
has no effect on their posttranscription (25). In contrast, polyamines
negatively regulate the stability of transforming growth factor-
mRNA without affecting the gene transcription (24). Clearly, further
studies are necessary to determine the transcriptional and
posttranscriptional regulation of the p53 gene in IEC-6 cells after
polyamine depletion.
In summary, these results indicate that inhibition of polyamine synthesis by treatment with DFMO significantly increases expression of the p53 gene in IEC-6 cells. Although the exact role of p53 protein in DFMO-treated cells is still unclear, our results clearly show that increased p53 gene expression after polyamine depletion does not induce programmed cell death. The association of induced p53 and a significant increase in G1 phase growth arrest in DFMO-treated cells suggests that the activation of p53 gene expression may play an important role in the process by which polyamine depletion results in growth inhibition.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. Leonard R. Johnson (The University of Tennessee College of Medicine) for helpful discussion and Dr. Diane C. Fadely for critical reading of the manuscript.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-45314 and a Merit Review Grant from the Department of Veterans Affairs to J.-Y. Wang.
Some of these data have been published in abstract form (32).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J.-Y. Wang, Dept. of Surgery, Baltimore VA Medical Center and University of Maryland, 10 North Greene St., Baltimore, MD 21201 (E-mail: jwang{at}surgery1.umaryland.edu).
Received 11 August 1998; accepted in final form 6 January 1999.
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