Expression of the transforming growth factor-beta gene during growth inhibition following polyamine depletion

Anami R. Patel, Ji Li, Barbara L. Bass, and Jian-Ying Wang

Department of Surgery, University of Maryland Medical School and Baltimore Veterans Affairs Medical Center, Baltimore, Maryland 21201

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Polyamine depletion and cytokine transforming growth factor-beta (TGF-beta ) inhibit cell proliferation. The current study tests the hypothesis that polyamine depletion results in growth inhibition by altering expression of the TGF-beta 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 alpha -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-beta . Increased TGF-beta mRNA in DFMO-treated cells was paralleled by an increase in TGF-beta content. Depletion of intracellular polyamines by DFMO had no effect on the rate of TGF-beta gene transcription, as measured by nuclear run-on assay. The half-life of mRNA for TGF-beta 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-beta mRNA in IEC-6 cells. TGF-beta 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-beta antibody. These results indicate that 1) depletion of intracellular polyamines induces the activation of the TGF-beta gene through posttranscriptional regulation and 2) increased expression of the TGF-beta gene plays an important role in the process of growth inhibition following polyamine depletion.

cell proliferation; intestinal crypt; posttranscription; ornithine decarboxylase

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-beta (TGF-beta ) 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-beta are found on nearly all cell types, but the nature of the biological response to TGF-beta varies with cell type (2). Although initially identified through its ability to stimulate cell proliferation in nontransformed fibroblasts, TGF-beta 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-beta inhibits cell proliferation and in some instances facilitates the development of differentiated function (1, 15). TGF-beta 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-beta 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-beta gene. To test this hypothesis, we first examined whether growth inhibition following polyamine depletion by alpha -difluoromethylornithine (DFMO), a specific inhibitor of ODC, is associated with an increased expression of the TGF-beta gene in IEC-6 cells, derived from rat small intestinal crypt cells. Second, we examined the role of TGF-beta mRNA synthesis and degradation in the observed activation of the TGF-beta gene in polyamine-deficient cells. Third, we examined cell proliferation rates when exogenous TGF-beta and anti-TGF-beta antibody were added to the cultures in the presence or absence of DFMO. Some of these data have been published in abstract form (29).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 pRTGFbeta 1 containing rat TGF-beta 1 cDNA [American Type Culture Collection (ATCC) no. 63197] and pHcGAP containing human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (ATCC no. OM-11-904A). [alpha -32P]dCTP (3,000 Ci/mmol) and [alpha -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-beta 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-beta gene expression in polyamine-deficient cells. Initially, we investigated the effect of polyamine depletion on the rate of TGF-beta 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-beta gene was measured by nuclear run-on transcription analysis. We then examined the effect of intracellular polyamines on the posttranscriptional regulation of TGF-beta mRNA. The half-life of TGF-beta 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-beta 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-beta or anti-TGF-beta antibody was added to the cultures. TGF-beta and immunoneutralizing anti-TGF-beta 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-beta or anti-TGF-beta 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-beta and GAPDH were labeled with [alpha -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 [alpha -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 [alpha -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-beta (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 [alpha -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-beta content. The level of TGF-beta in culture supernatants was measured with the use of the TGF-beta 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-beta 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-beta 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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of polyamine depletion on expression of the TGF-beta 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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Fig. 1.   Intracellular polyamine concentrations in IEC-6 cells grown in the presence or absence of 5 mM alpha -difluoromethylornithine (DFMO). Cells were grown in DMEM containing 5% dialyzed FBS. Medium was changed every 2nd day, and cellular polyamine levels were determined by HPLC analysis at various days after plating. Values are means ± SE of data from 6 dishes. * P < 0.05 compared with controls.

Depletion of cellular polyamines by treatment with DFMO significantly increased expression of the TGF-beta gene in IEC-6 cells (Fig. 2). The levels of TGF-beta 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-beta mRNA was paralleled by that of TGF-beta content (Fig. 2C). In the presence of DFMO, increased levels of TGF-beta mRNA and TGF-beta were completely prevented by addition of exogenous spermidine (5 µM). The levels of TGF-beta 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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Fig. 2.   Expression of transforming growth factor-beta (TGF-beta ) gene in control cells and cells treated with either DFMO or DFMO + spermidine (SPD). A: representative autoradiograms from control cells and cells exposed to DFMO or DFMO + SPD. Cells were grown in DMEM containing 5% dialyzed FBS in the presence or absence of DFMO or DFMO + SPD for 6 and 12 days. mRNA levels for TGF-beta were determined by Northern blotting using a TGF-beta 1 cDNA probe. B: quantitative analysis of Northern blots by densitometry from cells described in A. Relative levels of mRNA for TGF-beta were corrected for RNA loading as measured by densitometry of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). C: level of TGF-beta from cells described in A as measured by TGF-beta 1 ELISA system. Values are means ± SE of data from 3 separate experiments. * P < 0.05 compared with controls.

In normal cells grown without DFMO, the steady-state level of TGF-beta mRNA was not altered by long-term treatment with exogenous polyamines (data not shown). There were no significant differences in TGF-beta 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-beta gene. In this study, nuclear run-on transcription assay was employed to determine whether increases in steady-state levels of TGF-beta mRNA in the DFMO-treated cells resulted from an increased rate of TGF-beta gene transcription. As shown in Fig. 3, depletion of cellular polyamines by DFMO had no effect on TGF-beta gene transcription in the presence or absence of spermidine. There is no significant difference of the rates of TGF-beta 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-beta 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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Fig. 3.   Rate of TGF-beta gene transcription in response to polyamine depletion in IEC-6 cells. Cells were grown in control cultures and in cultures containing either DFMO (5 mM) or DFMO + 5 µM SPD for 6 days. Nuclei were isolated, and rate of transcription of TGF-beta gene was measured by nuclear run-on transcription analysis. Conditions for nuclear run-on transcription are described in MATERIALS AND METHODS. Equal amounts of [alpha -32P]UTP-labeled RNA (5 × 106 counts/min) were hybridized to filter containing immobilized plasmids of TGF-beta , pSV2-neo, and GAPDH. Three experiments were performed that showed similar results.

To examine the role of polyamines in posttranscriptional regulation of the TGF-beta gene, the possibility that polyamine depletion alters the stability of TGF-beta mRNA was determined in IEC-6 cells. We measured the half-life of TGF-beta 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-beta 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-beta mRNA in polyamine-deficient cells was dramatically increased. TGF-beta 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-beta mRNA in the DFMO-treated cells was prevented when spermidine was given together with DFMO. The half-life of mRNA for TGF-beta was at a normal level in the cells exposed to DFMO plus spermidine for 6 days. The TGF-beta 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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Fig. 4.   Cytoplasmic half-life studies of TGF-beta mRNA from control cells (A) and cells treated with either DFMO (B) or DFMO + SPD (C). Cells were untreated or treated with DFMO or DFMO + SPD for 6 days and incubated further with 5 µg actinomycin D/ml for indicated times. Total RNA was analyzed by Northern blotting using a TGF-beta 1 cDNA probe. Loading of RNA was monitored by hybridization to labeled GAPDH probe. Three experiments were performed that showed similar results.


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Fig. 5.   Percent of TGF-beta mRNA remaining in IEC-6 cells described in Fig. 4. Values are means from 3 separate experiments, and relative levels of TGF-beta mRNA were corrected for RNA loading as measured by densitometry of GAPDH.

Effect of polyamine depletion on cell proliferation. Consistent with the effect on expression of the TGF-beta 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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Fig. 6.   DNA synthesis (A) and cell number (B) in IEC-6 cells exposed to either 5 mM DFMO or DFMO + 5 µM SPD. Cells were grown in DMEM containing 5% dialyzed FBS in the presence or absence of DFMO or DFMO + SPD for 6 and 12 days. Medium was changed every 2nd day, and DNA synthesis, as measured by [3H]thymidine incorporation technique, and cell number were determined at 6 and 12 days after initial plating. Values are means ± SE of data from 6 dishes. * P < 0.05 compared with controls (Con); cpm, counts/min.

Increased TGF-beta in growth inhibition following polyamine depletion. To elucidate whether increased expression of the TGF-beta gene is involved in the process of growth inhibition after polyamine depletion, we first examined the effect on IEC-6 cell proliferation when TGF-beta was added to control cultures and cultures containing 5 mM DFMO. Figure 7 clearly shows that exposure to TGF-beta 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-beta 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-beta 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-beta increased the sensitivity of TGF-beta inhibition significantly. In DFMO-treated cells, TGF-beta at a dose of 2.5 ng/ml significantly decreased DNA synthesis and cell number. When TGF-beta 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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Fig. 7.   Effect of exogenous TGF-beta added to standard culture medium (without DFMO) on DNA synthesis (A) and cell number (B) in IEC-6 cells. Cells were grown in DMEM containing 5% dialyzed FBS for 4 days, and then TGF-beta was given at different concentrations. DNA synthesis and cell number were assayed 48 h after administration of TGF-beta . Values are means ± SE of data from 6 dishes. * P < 0.05 compared with controls.


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Fig. 8.   Effect of exogenous TGF-beta added to cultures containing DFMO on DNA synthesis (A) and cell number (B) in IEC-6 cells. Cells were grown in DMEM containing 5% dialyzed FBS in the presence of DFMO for 4 days, and then TGF-beta was given at different concentrations. DNA synthesis and cell number were assayed 48 h after administration of TGF-beta in the presence of DFMO. Values are means ± SE of data from 6 dishes. * P < 0.05 compared with controls.

Second, we examined whether anti-TGF-beta 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-beta 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-beta 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-beta added to the culture medium in polyamine-deficient cells (Fig. 9, B and D). In the presence of exogenous TGF-beta (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-beta 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-beta (data not shown). These results strongly suggest that increased expression of the TGF-beta gene plays an important role in growth inhibition caused by polyamine depletion.


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Fig. 9.   Effect of immunoneutralizing anti-TGF-beta antibody added to cultures containing DFMO on DNA synthesis (A and B) and cell number (C and D) in IEC-6 cells. A: DNA synthesis in cells exposed to DFMO and anti-TGF-beta antibody. Cells were pretreated with DFMO for 4 days and then incubated with antibody against TGF-beta at different concentrations for additional 48 h in the presence of DFMO. DNA synthesis was assayed by [3H]thymidine incorporation technique. B: ability of antibody to immunoneutralize exogenous TGF-beta added to cultures containing DFMO. After cells were exposed to DFMO for 4 days, TGF-beta alone or TGF-beta  + antibody were given. DNA synthesis was measured 48 h after treatments. C: cell number from cultures described in A. D: cell number from cultures described in B. Values are means ± SE of data from 6 dishes. * P < 0.05 compared with controls. + P < 0.05 compared with TGF-beta alone.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-beta mRNA in IEC-6 cells (Fig. 2). Increased levels of TGF-beta mRNA in DFMO-treated cells were paralleled by an increase in TGF-beta content. Although polyamine depletion has no effect on the transcription of the TGF-beta 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-beta antibody to the culture medium partially but significantly prevents growth inhibition in the DFMO-treated cells (Fig. 9), suggesting that activation of the TGF-beta 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-beta is an important negative regulator of cell proliferation in a wide variety of cell types including intestinal epithelial cells (1, 21). Administration of TGF-beta inhibits proliferative activity and promotes the development of differentiated function in IEC-6 cells (15). The anti-proliferative action of TGF-beta is not mediated through its ability to antagonize the mitogenic effects of other peptides because inhibition of IEC-6 cells by TGF-beta 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-beta 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-beta mRNA, which is paralleled by an increase in TGF-beta content. Because the increase in the levels of TGF-beta 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-beta 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-beta 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-beta 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-beta 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-beta 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-beta mRNA levels more than twofold of control values (Fig. 2). These results indicate that the increased expression of the TGF-beta 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-beta mRNA but has no effect on the rate of TGF-beta gene transcription. These findings suggest that intracellular polyamines play a major role in the posttranscriptional regulation of the TGF-beta gene and that the increase in steady-state levels of TGF-beta 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-beta 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-beta 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-beta 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-beta gene expression to wounding in the DFMO-treated cells with that observed in controls (without DFMO). The levels of TGF-beta and its mRNA were measured at various times after wounding. The current study examines the relationship between basal expression of the TGF-beta 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-beta 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-beta 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-beta mRNA but has no effect on the basal rate of TGF-beta gene transcription.

Increased expression of the TGF-beta 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-beta 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-beta also increased the sensitivity to inhibitory effects of TGF-beta on cell growth. However, the functional importance of increased TGF-beta following polyamine depletion is shown by the ability of immunoneutralizing anti-TGF-beta 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-beta antibody for 48 h. Although the mechanism by which TGF-beta inhibits cell growth in intestinal mucosal tissue remains obscure, several studies have shown that TGF-beta has only a small effect on differentiation and does not induce terminal differentiation (1, 15), suggesting that the inhibitory effect of TGF-beta 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-beta 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-beta mRNA occur on day 6 after DFMO treatment (28). It also remains to be elucidated that activity at the TGF-beta receptor is essential to growth inhibition in polyamine-deficient cells. Clearly, further studies are necessary to determine the exact role of increased TGF-beta 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-beta gene in normal small intestinal crypt cells. These results also show that polyamine depletion dramatically increases the half-life of TGF-beta mRNA but has no effect on the rate of transcription of the TGF-beta gene. It is indicated that cellular polyamines play a critical role in the regulation of posttranscription of the TGF-beta gene and that increased levels of TGF-beta 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-beta antibody to the culture medium, increased expression of the TGF-beta gene plays an important role in the process of growth inhibition following polyamine depletion.

    ACKNOWLEDGEMENTS

We thank Dr. Diane C. Fadely for critical reading of the manuscript and Jordan Denner (Baltimore Veterans Affairs Medical Center) for illustrations.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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