Department of Surgery, University of Maryland Medical School and Baltimore Veterans Affairs Medical Center, Baltimore, Maryland 21201
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ABSTRACT |
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Polyamine depletion and cytokine transforming growth factor-
(TGF-
) inhibit cell proliferation. The current study tests the
hypothesis that polyamine depletion results in growth inhibition by
altering expression of the TGF-
gene in intestinal epithelial cells.
Studies were conducted in the IEC-6 cell line derived from rat small
intestinal crypt cells. Cells were grown in DMEM in the presence or
absence of
-difluoromethylornithine (DFMO), a specific inhibitor of
polyamine biosynthesis, for 6 and 12 days. Administration of DFMO not
only depleted intracellular polyamines but also significantly increased
the mRNA levels of TGF-
. Increased TGF-
mRNA in DFMO-treated
cells was paralleled by an increase in TGF-
content. Depletion of
intracellular polyamines by DFMO had no effect on the rate of TGF-
gene transcription, as measured by nuclear run-on assay. The half-life
of mRNA for TGF-
in normal cells was ~65 min and increased to
>16 h in cells treated with DFMO for 6 or 12 days. Exogenous
polyamine, when given together with DFMO, prevented the increased
half-life of TGF-
mRNA in IEC-6 cells. TGF-
added to the culture
medium significantly decreased the rate of DNA synthesis and final cell
number in normal and polyamine-deficient cells. Furthermore, growth
inhibition caused by polyamine depletion was partially but
significantly blocked by addition of immunoneutralizing anti-TGF-
antibody. These results indicate that
1) depletion of intracellular
polyamines induces the activation of the TGF-
gene through
posttranscriptional regulation and
2) increased expression of the
TGF-
gene plays an important role in the process of growth
inhibition following polyamine depletion.
cell proliferation; intestinal crypt; posttranscription; ornithine decarboxylase
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INTRODUCTION |
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THE EPITHELIUM OF THE intestinal mucosa has the most rapid turnover rate of any tissue in the body and is continuously renewed from the proliferative zone of undifferentiated stem cells within the crypts (12, 16). Mature differentiated cells sloughed into the lumen from the villous tip are quickly replaced by cell proliferation. The normal structure and function of the tissue depend on a regulated rate of division of proliferating mucosal crypt cells (12, 16). Our previous studies have demonstrated that the polyamines spermidine and spermine and their precursor, putrescine, are absolutely required for cell proliferation in the crypts of the small intestinal mucosa and that intracellular polyamine levels are highly regulated by the cells according to the state of growth (18, 30). Decreasing cellular polyamines by inhibition of the activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, inhibits cell renewal in intestinal mucosal tissue (17, 26, 33). However, the precise mechanism of mucosal growth inhibition following polyamine depletion at the molecular level has not been elucidated.
The transforming growth factor- (TGF-
) family consists of a group
of closely related genes and is widely distributed in a variety of
human and animal tissues (2, 3). The receptors for TGF-
are found on
nearly all cell types, but the nature of the biological response to
TGF-
varies with cell type (2). Although initially identified
through its ability to stimulate cell proliferation in nontransformed
fibroblasts, TGF-
was later shown to be a potent growth inhibitor
for a wide variety of cell types (2, 21). Studies on the epithelial
cells of the intestinal mucosa have indicated that TGF-
inhibits
cell proliferation and in some instances facilitates the development of
differentiated function (1, 15). TGF-
also exerts many other
biological effects in the gut mucosa, including regulation of cell
adhesion and migration, stimulation of extracellular matrix production, and modulation of immune and endocrine functions (2, 4).
Because polyamine depletion and TGF- inhibit cell proliferation, it
is reasonable to consider the possibility that depletion of
intracellular polyamines suppresses intestinal epithelial cell growth
by altering expression of the TGF-
gene. To test this hypothesis, we
first examined whether growth inhibition following polyamine depletion
by
-difluoromethylornithine (DFMO), a specific inhibitor of ODC, is
associated with an increased expression of the TGF-
gene in IEC-6
cells, derived from rat small intestinal crypt cells. Second, we
examined the role of TGF-
mRNA synthesis and degradation in the
observed activation of the TGF-
gene in polyamine-deficient cells.
Third, we examined cell proliferation rates when exogenous TGF-
and
anti-TGF-
antibody were added to the cultures in the presence or
absence of DFMO. Some of these data have been published in abstract
form (29).
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MATERIALS AND METHODS |
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Materials. Disposable culture ware was
purchased from Corning Glass Works (Corning, NY). Tissue culture media
and dialyzed FBS were from GIBCO (Grand Island, NY). Biochemicals were
purchased from Sigma (St. Louis, MO). The DNA probes used in these
experiments included pRTGF1 containing rat TGF-
1 cDNA
[American Type Culture Collection (ATCC) no. 63197] and
pHcGAP containing human glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) cDNA (ATCC no. OM-11-904A).
[
-32P]dCTP (3,000 Ci/mmol) and
[
-32P]UTP (800 Ci/mmol) were purchased from Amersham (Arlington Heights, IL) and
[3H]thymidine (2 Ci/mmol) was from New England Nuclear (Boston, MA). DFMO was a gift
from the Merrell Dow Research Institute of Marion Merrell Dow
(Cincinnati, OH).
Cell culture and general experimental protocol. The IEC-6 cell line was purchased from the ATCC at passage 13. The cell line was derived from normal rat intestine and was developed and characterized by Quaroni et al. (23). 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, 10 µg 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, and medium was changed three times per week. 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 (28).
The general protocol of the experiments and the methods used were similar to those described previously (32). Briefly, IEC-6 cells were plated at 6.25 × 104 cells/cm2 in DMEM + 5% dialyzed FBS + 10 µg insulin and 50 µg gentamicin sulfate/ml (supplemented DMEM). They were incubated in a humidified atmosphere at 37°C in 90% air-10% CO2 for 24 h, which was then followed by a period of different experimental treatments.
In the first series of studies, we investigated whether growth
inhibition produced by polyamine depletion is associated with an
increased expression of the TGF- gene in IEC-6 cells. Cells were
grown in control cultures, in cultures containing 5 mM DFMO, and in
cultures with DFMO + 5 µM spermidine for 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 possible mechanisms
responsible for the observed activation of TGF- gene expression in
polyamine-deficient cells. Initially, we investigated the effect of
polyamine depletion on the rate of TGF-
gene transcription in IEC-6
cells. After cells were exposed to DFMO for 6 and 12 days, their nuclei
were isolated, and the rate of transcription of the TGF-
gene was
measured by nuclear run-on transcription analysis. We then examined the
effect of intracellular polyamines on the posttranscriptional
regulation of TGF-
mRNA. The half-life of TGF-
mRNA was measured
in control cultures, in cultures containing DFMO, and in cultures with
DFMO plus spermidine. Actinomycin D (5 µg/ml) was added to the
cultures after cells were grown in the presence or absence of DFMO for
6 or 12 days. The levels of TGF-
mRNA were assayed at different
times after the addition of actinomycin D.
In the third series of studies, we examined cell proliferation rates in
normal and polyamine-deficient IEC-6 cells when exogenous TGF- or
anti-TGF-
antibody was added to the cultures. TGF-
and
immunoneutralizing anti-TGF-
antibody at different concentrations were added to control cultures and to cultures in which cellular polyamines had been depleted by treatment with 5 mM DFMO for 4 days.
The rate of DNA synthesis and cell number were assayed 48 h after
administration of either TGF-
or anti-TGF-
antibody in the
presence or absence of DFMO.
RNA isolation and Northern blot
analysis. Total RNA was extracted with guanidinum
isothiocyanate solution and purified by CsCl density gradient
ultracentrifugation as described by Chirgwin et al. (7). Briefly, the
monolayer of cells was washed in D-PBS and lysed in 4 M guanidinium
isothiocyanate. The lysates were 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 in sequence of 0.1 vol of 3 M sodium acetate and
2.5 vol of ethanol. Final RNA was dissolved in water and estimated from
its ultraviolet absorbance at 260 nm using a conversion factor of 40 units. In most cases, 30 µg of total cellular RNA were denatured and
fractionated electrophoretically using a 1.2% agarose gel containing
3% formaldehyde and were transferred by blotting to nitrocellulose
filters. Blots were prehybridized for 24 h at 42°C with 5×
Denhardt's solution-5× standard saline salmon sperm DNA. DNA
probes for TGF- 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× standard sodium
citrate (SSC)/0.1% SDS for 10 min 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 autoradiographic results.
Nuclear run-on assays. Nuclei were
prepared according to established methods of deBustros et al. (9).
Briefly, IEC-6 cells were suspended in buffer
A (in mM: 20 Tris · HCl, pH 7.4, 10 NaCl, and 3 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 at
1,000 g 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 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 (20). 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 (17) as described in
RNA isolation and Northern blot
analysis.
Immobilization of DNA plasmids and
hybridization. The cDNAs for TGF-
(BamH I), GAPDH
(Hind III), and pSV2-neo
were linearized by digestion with the respective enzymes and were
boiled and blotted (20 µg of cDNA/blot) onto nitrocellulose membrane
(Gene Screen; Du Pont). The GAPDH served as a positive control and
pSV2-neo as a negative control. After these membranes were dried at
80°C for 2 h, they were prehybridized overnight and then hybridized with [
-32P]RNA
isolated from nuclear transcription experiments for 24 h at 42°C.
Filters were washed in 2× SSC, 1% SDS at 65°C for 1 h and
then in 0.1× SSC, 0.1% SDS at room temperature for 1 h. Filters were exposed to Kodak XAR-2 film at
70°C. Quantitative
results were obtained by densitometric scanning and are expressed with reference to the signal for GAPDH.
Measurement of TGF-
content. The level of TGF-
in culture supernatants
was measured with the use of the TGF-
1 ELISA system (Promega,
Madison, WI). After cells in 30-mm dishes were grown in the presence or
absence of DFMO for 6 and 12 days, the monolayer of cells was washed
once with D-PBS, and then 1 ml of fresh medium was added. The medium
was collected following 12 h of further culture, and the content of
TGF-
was measured according to the manufacture's instructions.
Cells were dissolved in 0.5 ml of 0.5 N NaOH at 37°C in humidified
air for 90 min. The protein content of an aliquot of cell lysate was
determined by the method described by Bradford (5). The level of
TGF-
content was normalized by protein and expressed as picograms
per milliliter per milligram of protein.
Measurement of DNA synthesis. DNA synthesis was measured with the use of the [3H]thymidine incorporation technique as previously described (14). This method has been validated as a measure of DNA synthesis in IEC-6 cells by means of specific DNA polymerase inhibitor (10). Cells in 24-well plates were pulsed with 1 µCi/ml of [3H]thymidine for 4 h before harvest. Cells were washed twice with cold D-PBS solution and were then incubated in cold 10% TCA for 30 min at 4°C. After rinsing twice with cold 10% TCA, the 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 described by Bradford (5). DNA synthesis was expressed as disintegrations per minute per microgram of protein.
Polyamine analysis. The cellular
polyamine content was analyzed by HPLC as described previously (24). In
brief, after washing the monolayers three times with ice-cold D-PBS, we
added 0.5 M perchloric acid then froze the monolayers at
80°C until they were ready for extraction, dansylation, and
HPLC. The standard curve encompassed 0.31-10 µM putrescine,
spermidine, and spermine. Values that fell >25% below the curve were
considered undetectable. Protein was determined by the Bradford method
(5). The amount of polyamines was expressed as nanomoles per milligram
of protein.
Statistics. All data are expressed as 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 the Dunnett's multiple-range test (11), and values of P < 0.05 were considered significant.
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RESULTS |
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Effect of polyamine depletion on expression of the
TGF- gene. The exposure of IEC-6 cells
to 5 mM DFMO totally inhibited ODC activity and almost completely
depleted the cellular polyamines. As can be seen in Fig.
1, administration of 5 mM DFMO for 6 and 12 days decreased intracellular putrescine and spermidine content to
undetectable levels. Spermine was less sensitive to the inhibition of
ODC and was decreased by >50% on day
6 and by 80% on day
12.
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Depletion of cellular polyamines by treatment with DFMO significantly
increased expression of the TGF- gene in IEC-6 cells (Fig.
2). The levels of TGF-
mRNA in the cells
treated with DFMO for 6 and 12 days were approximately two times the
normal value (without DFMO; Fig. 2, A
and B). The change in TGF-
mRNA
was paralleled by that of TGF-
content (Fig.
2C). In the presence of DFMO,
increased levels of TGF-
mRNA and TGF-
were completely prevented
by addition of exogenous spermidine (5 µM). The levels of TGF-
and
its mRNA in cells grown in the presence of DFMO plus spermidine for 6 and 12 days were indistinguishable from those of control cells.
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In normal cells grown without DFMO, the steady-state level of TGF-
mRNA was not altered by long-term treatment with exogenous polyamines
(data not shown). There were no significant differences in TGF-
mRNA
levels between control cells and cells exposed to putrescine (10 µM)
or spermidine (5 µM) for 12 days.
Effect of polyamine depletion on transcription and
posttranscription of the TGF- gene. In
this study, nuclear run-on transcription assay was employed to
determine whether increases in steady-state levels of TGF-
mRNA in
the DFMO-treated cells resulted from an increased rate of TGF-
gene
transcription. As shown in Fig. 3, depletion of cellular polyamines by DFMO had no effect on TGF-
gene
transcription in the presence or absence of spermidine. There is no
significant difference of the rates of TGF-
gene transcription between control cells and cells exposed to either DFMO or DFMO plus
spermidine for 6 days. We also measured the rate of TGF-
gene
transcription in the cells treated with DFMO for 12 days and
demonstrated that the results were identical to those observed after
treatment for 6 days.
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To examine the role of polyamines in posttranscriptional regulation of
the TGF- gene, the possibility that polyamine depletion alters the
stability of TGF-
mRNA was determined in IEC-6 cells. We measured
the half-life of TGF-
mRNA in cells grown in the presence or absence
of DFMO for 6 and 12 days. As shown in Figs. 4 and 5, the
mRNA levels for TGF-
in control cells declined rapidly after the
administration of actinomycin D, with a half-life of 65 min. In the
DFMO-treated cells, however, the stability of TGF-
mRNA in
polyamine-deficient cells was dramatically increased. TGF-
mRNA from
cells exposed to DFMO for 6 days decreased at a slower rate, with a
half-life of >16 h. The increased stability of TGF-
mRNA in the
DFMO-treated cells was prevented when spermidine was given together
with DFMO. The half-life of mRNA for TGF-
was at a normal
level in the cells exposed to DFMO plus spermidine for 6 days. The TGF-
mRNA half-life of the cells exposed to DFMO for 12 days was identical to that observed after a 6-day treatment (data not
shown).
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Effect of polyamine depletion on cell
proliferation. Consistent with the effect on expression
of the TGF- gene, polyamine depletion significantly decreased cell
proliferation. The rate of DNA synthesis and cell number in the
DFMO-treated cells were markedly decreased (Fig.
6). The administration of spermidine (5 µM) reversed the inhibitory effects of DFMO on cell proliferation. The reduced rate of DNA synthesis and cell number in cells treated with
DFMO returned toward control levels when spermidine was given.
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Increased TGF- in growth inhibition
following polyamine depletion. To elucidate whether
increased expression of the TGF-
gene is involved in the process of
growth inhibition after polyamine depletion, we first examined the
effect on IEC-6 cell proliferation when TGF-
was added to control
cultures and cultures containing 5 mM DFMO. Figure
7 clearly shows that exposure to TGF-
for 48 h significantly inhibited the rate of DNA synthesis and final cell number in cells grown in the standard culture medium (without DFMO). When various doses of TGF-
were tested, the dose of 1 ng/ml
slightly increased DNA synthesis and cell number, but these differences
were not statistically significant. Cell proliferation was inhibited
linearly with concentrations of TGF-
ranging from 2.5 to 10 ng/ml.
Significant decreases in DNA synthesis and cell number occurred first
at 10 ng/ml and were ~55% of normal values. As can be seen in Fig.
8, polyamine depletion before the addition of TGF-
increased the sensitivity of TGF-
inhibition
significantly. In DFMO-treated cells, TGF-
at a dose of 2.5 ng/ml
significantly decreased DNA synthesis and cell number. When TGF-
at
a dose of 5 or 10 ng/ml was given, DNA synthesis and cell number were ~20% of control values and were decreased by >70%.
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Second, we examined whether anti-TGF- antibody added to the cultures
containing DFMO promoted cell proliferation in polyamine-deficient cells. Cells were grown in the presence of DFMO for 4 days, and then
antibody against TGF-
at different concentrations was added to the
medium containing DFMO. DNA synthesis and cell number were measured
after incubation with the antibody for 48 h. Administration of
anti-TGF-
antibody resulted in a significant induction of DNA
synthesis (Fig.
9A) and
final cell number (Fig. 9C) in the DFMO-treated cells. The rate of
[3H]thymidine
incorporation and cell number in the DFMO-treated cells were increased
by 70 and 60%, respectively, when the antibody at the concentration of
20 µg/ml was given. Although the antibody at the dose of 30 µg/ml
also increased cell division, the values reached were no higher than
the response to 20 µg/ml (data not shown). This antibody also has the
ability to immunoneutralize exogenous TGF-
added to the culture
medium in polyamine-deficient cells (Fig. 9,
B and
D). In the presence of exogenous
TGF-
(5 ng/ml), the maximum increase in DNA synthesis and cell
number in the DFMO-treated cells occurred after exposure to the
antibody at the dose of 30 µg/ml. We also tested the effect of
heat-inactivated anti-TGF-
antibody on cell proliferation in the
DFMO-treated cells and demonstrated that the nonbinding antibody had no
additional effects on the rate of DNA synthesis and cell number,
regardless of the presence or absence of exogenous TGF-
(data not
shown). These results strongly suggest that increased expression of the TGF-
gene plays an important role in growth inhibition caused by
polyamine depletion.
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DISCUSSION |
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As pointed out in the introduction, an adequate supply of polyamines in
the dividing cells within the crypts is absolutely required for small
intestinal epithelial cell proliferation. Inhibition of polyamine
synthesis significantly suppresses mucosal growth (27, 28), but the
mechanism of growth inhibition remains to be demonstrated. The current
study clearly shows that polyamine depletion by treatment with DFMO is
associated with an increase in the level of TGF- mRNA in IEC-6 cells
(Fig. 2). Increased levels of TGF-
mRNA in DFMO-treated cells were
paralleled by an increase in TGF-
content. Although polyamine
depletion has no effect on the transcription of the TGF-
gene (Fig.
3), the half-life of mRNA is dramatically increased in cells grown in the presence of DFMO for 6 and 12 days (Figs. 4 and 5). Furthermore, addition of immunoneutralizing anti-TGF-
antibody to the culture medium partially but significantly prevents growth inhibition in the
DFMO-treated cells (Fig. 9), suggesting that activation of the TGF-
gene is one mechanism by which decreasing cellular polyamine levels
inhibits cell renewal in the intestinal mucosa.
In normal mammalian tissue, a balance between proliferation, growth arrest, and apoptosis regulates cell numbers. A series of observations from our previous studies (22, 28, 30) and others (6) has demonstrated that polyamines are essential for the stimulation of intestinal epithelial cell proliferation at least partially in association with their ability to modulate protooncogene expression. It has been shown that induction of cell division in vivo (25) as well as in vitro (28) is associated with a significant increase in the expression of protooncogenes following increased polyamine synthesis, which precedes the induction of DNA synthesis. Inhibition of polyamine synthesis prevents increases in both protooncogene expression and cell division. These results indicate that increased expression of protooncogenes is involved in the early modulation of mucosal growth and may be part of the mechanism responsible for polyamine-stimulated cell division. Because mucosal epithelial cells continue to maintain a high basal level of protooncogene expression after inhibition of polyamine synthesis (28, 32), growth arrest following polyamine depletion may result from a mechanism other than a simple decrease in protooncogene expression. The change in activation of protooncogene expression plays a minor role in growth inhibition of intestinal crypt cells following polyamine depletion, although it is absolutely required for the stimulation of cell proliferation by polyamines.
The recognition that negative growth control must be elucidated to
comprehend the mechanisms by which appropriate cell numbers are
maintained has attracted considerable interest. In the small intestinal
mucosa, mitotic activity is confined to the crypt region and must be
finely tuned to the rapid rate of loss of mature enterocytes from the
villus. Normal division of crypt cells is regulated by negative
control, with suppression of cell renewal mediated through a factor
produced in the villus (12, 16). It has been shown that TGF- is an
important negative regulator of cell proliferation in a wide variety of
cell types including intestinal epithelial cells (1, 21).
Administration of TGF-
inhibits proliferative activity and promotes
the development of differentiated function in IEC-6 cells (15). The
anti-proliferative action of TGF-
is not mediated through its
ability to antagonize the mitogenic effects of other peptides because
inhibition of IEC-6 cells by TGF-
is observed in the absence of any
additional growth factors and is not diminished by other growth
factors.
The findings reported here indicate that expression of the TGF- gene
is involved in the mechanism by which polyamine depletion results in
growth inhibition of intestinal epithelial cells. As noted in Fig. 2,
administration of DFMO for 6 and 12 days is associated with increased
levels for TGF-
mRNA, which is paralleled by an increase in TGF-
content. Because the increase in the levels of TGF-
and its mRNA in
DFMO-treated cells is completely prevented by the addition of exogenous
spermidine, these observed changes in the activation of the TGF-
gene must have been related to polyamine depletion rather than to the
nonspecific effect of DFMO. Our results also indicate that long-term
administration of putrescine or spermidine for 12 days has no
inhibitory effect on basal levels of TGF-
mRNA when cells are grown
in a standard culture medium containing no DFMO (data not shown). These
results are consistent with our previous findings (28) that none of the
polyamines are able to stimulate cell proliferation over normal levels
when added to control cells.
It is necessary to distinguish those events that were a result of
inhibited growth from those induced by polyamine depletion to elucidate
the relationship between decreasing cellular polyamines and the
activation of TGF- gene expression in DFMO-treated cells. In the
current study, we compared polyamine-deficient cells with control cells
that were growth inhibited at confluence. Our previous studies (28)
have demonstrated that confluent growth inhibition of IEC-6 cells is
not associated with a significant decrease in cellular polyamines. As
can be seen in Fig. 6, both controls and cells exposed to DFMO with or
without spermidine entered a plateau phase by day
6 after initial plating, and there was no additional increase in cell number between days 6 and 12 in these three groups. Assays
were carried out during growth inhibition produced by either cellular
confluence or polyamine depletion. The expression of the TGF-
gene
in the controls can then be compared with effects resulting from the
depletion of polyamines by DFMO. There were low basal levels of TGF-
mRNA on days 6 and
12 in control cells and cells exposed
to DFMO plus spermidine, both of which were growth inhibited at
confluence. However, polyamine depletion increased TGF-
mRNA levels
more than twofold of control values (Fig. 2). These results indicate
that the increased expression of the TGF-
gene in the DFMO-treated
cells is related to polyamine depletion and is not the result of
decreased growth.
Figures 3 and 4 show that administration of DFMO for 6 or 12 days
dramatically increases the half-life of TGF- mRNA but has no effect
on the rate of TGF-
gene transcription. These findings suggest that
intracellular polyamines play a major role in the posttranscriptional
regulation of the TGF-
gene and that the increase in steady-state
levels of TGF-
mRNA in the DFMO-treated IEC-6 cells is primarily
caused by the decrease in mRNA degradation. It is not clear at present
whether prolonged stability of TGF-
mRNA following polyamine
depletion is mediated by other factors. Although the exact mechanism
responsible for the process of mRNA turnover in general is poorly
understood, increasing evidence suggests that the maintenance of mRNA
stability is related to the destabilizing sequences in the
3'-untranslated regions of mRNAs. Many transiently expressed
genes such as c-fos,
c-sis, c-myc, and ODC have been demonstrated
to contain destabilizing sequences (13, 19). Whether there are
destabilizing sequences in the 3'-untranslated regions of TGF-
mRNA and whether the action of polyamines involves this sequence in
IEC-6 cells remain to be elucidated.
We have recently reported that increased expression of the TGF- gene
during cell migration requires polyamines in IEC-6 cells (31). That
result is not incongruent with the current observation, because the
experimental conditions are completely different from those of our
current study. In the cell migration study, we compared the pattern of
rapid response of TGF-
gene expression to wounding in the
DFMO-treated cells with that observed in controls (without DFMO). The
levels of TGF-
and its mRNA were measured at various times after
wounding. The current study examines the relationship between basal
expression of the TGF-
gene and growth inhibition in cells exposed
to DFMO for 6 and 12 days without wounding. Of greatest importance,
however, is the finding that polyamine depletion alters expression of
the TGF-
gene during migration and growth inhibition through
different pathways. We have demonstrated that inhibition of polyamine
synthesis significantly prevents increases in the rate of TGF-
gene
transcription after wounding (data not published). It should be noted
that the studies in this report show that exposure to DFMO for 6 and 12 days dramatically increases the half-life of TGF-
mRNA but has no
effect on the basal rate of TGF-
gene transcription.
Increased expression of the TGF- gene in DFMO-treated cells plays a
major role in the process of growth inhibition caused by polyamine
depletion. As shown in Figs. 7 and 8, administration of exogenous
TGF-
significantly inhibits DNA synthesis and final cell number in
both control and DFMO-treated cells. Depletion of cellular polyamines
by DFMO before the addition of TGF-
also increased the sensitivity
to inhibitory effects of TGF-
on cell growth. However, the
functional importance of increased TGF-
following polyamine
depletion is shown by the ability of immunoneutralizing anti-TGF-
antibody to promote cell division in the presence of DFMO (Fig. 9). The
rate of [3H]thymidine
incorporation and cell number in the DFMO-treated cells are
significantly increased after incubation with anti-TGF-
antibody for
48 h. Although the mechanism by which TGF-
inhibits cell growth in
intestinal mucosal tissue remains obscure, several studies have shown
that TGF-
has only a small effect on differentiation and does not
induce terminal differentiation (1, 15), suggesting that the inhibitory
effect of TGF-
on cell proliferation in DFMO-treated IEC-6 cells
does not result from differentiation. In support of this possibility,
our previous studies have revealed no biochemical changes indicative of
differentiation after up to 12 days of DFMO treatment as evidenced by
lactase, maltase, and sucrase activity (28). In addition, the action of
TGF-
on the DFMO-treated cells is not due to its inhibitory effect
on the expression of the c-myc gene
(8), since decreased expression of
c-myc occurs on day 4, whereas increases in TGF-
mRNA occur on
day 6 after DFMO treatment (28). It
also remains to be elucidated that activity at the TGF-
receptor is
essential to growth inhibition in polyamine-deficient cells. Clearly,
further studies are necessary to determine the exact role of increased
TGF-
in the process of growth inhibition in polyamine-deficient
cells.
In summary, these results indicate that depletion of intracellular
polyamines by long-term treatment with DFMO is associated with the
increased expression of the TGF- gene in normal small intestinal
crypt cells. These results also show that polyamine depletion
dramatically increases the half-life of TGF-
mRNA but has no effect
on the rate of transcription of the TGF-
gene. It is indicated that
cellular polyamines play a critical role in the regulation of
posttranscription of the TGF-
gene and that increased levels of
TGF-
mRNA in polyamine-deficient cells primarily result from a delay
in the rate of mRNA degradation. Because decreased cell division in the
DFMO-treated cells is significantly blocked by addition of
immunoneutralizing anti-TGF-
antibody to the culture medium,
increased expression of the TGF-
gene plays an important role in the
process of growth inhibition following polyamine depletion.
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ACKNOWLEDGEMENTS |
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We thank Dr. Diane C. Fadely for critical reading of the manuscript and Jordan Denner (Baltimore Veterans Affairs Medical Center) for illustrations.
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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 by a Merit Review Grant from the Department of Veterans Affairs (to J.-Y. Wang).
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: J.-Y. Wang, Dept. of Surgery, Baltimore Veterans Affairs Medical Center and Univ. of Maryland, 10-North Greene St., Baltimore, MD 21201.
Received 27 January 1998; accepted in final form 30 April 1998.
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