Polyamine-modulated expression of c-myc plays a critical role in stimulation of normal intestinal epithelial cell proliferation

Lan Liu,1,3,* Li Li,1,3,* Jaladanki N. Rao,1,3 Tongtong Zou,1,3 Huifang M. Zhang,1,3 Dessy Boneva,1,3 Marasa S. Bernard,2,3 and Jian-Ying Wang1,2,3

Departments of 1Surgery and 2Pathology, University of Maryland School of Medicine, and 3Baltimore Veterans Affairs Medical Center, Baltimore, Maryland 21201

Submitted 8 July 2004 ; accepted in final form 1 September 2004


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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The nuclear protein c-Myc is a transcription factor involved in the control of cell cycle. Our previous studies indicated that cellular polyamines are absolutely required for cell proliferation in crypts of small intestinal mucosa and that polyamines have the ability to stimulate expression of the c-myc gene. The current study went further to determine whether induced nuclear c-Myc plays a role in stimulation of cell proliferation by polyamines in intestinal crypt cells (IEC-6 line). Exposure of normal quiescent cells after 24-h serum deprivation to 5% dialyzed fetal bovine serum (dFBS) increased both cellular polyamines and expression of the c-myc gene. Increased c-Myc protein formed heterodimers with its binding partner, Max, and specifically bound to the Myc/Max binding site, which was associated with an increase in DNA synthesis. Depletion of cellular polyamines by pretreatment with {alpha}-difluoromethylornithine (DFMO) prevented increases in c-myc expression and DNA synthesis induced by 5% dFBS. c-Myc gene transcription and cell proliferation decreased in polyamine-deficient cells, whereas the natural polyamine spermidine given together with DFMO maintained c-myc gene expression and cell growth at normal levels. Disruption of c-myc expression using specific c-myc antisense oligomers not only inhibited normal cell growth (without DFMO) but also prevented the restoration of cell proliferation by spermidine in polyamine-deficient cells. Ectopic expression of wild-type c-myc by recombinant adenoviral vector containing c-myc cDNA increased cell growth. These results indicate that polyamine-induced nuclear c-Myc interacts with Max, binds to the specific DNA sequence, and plays an important role in stimulation of normal intestinal epithelial cell proliferation.

ornithine decarboxylase; {alpha}-difluoromethylornithine; growth arrest; gene expression; intestinal epithelium


THE C-MYC GENE encodes a nuclear transcription factor that is involved in a variety of cellular functions (1, 4, 16, 21, 31). Myc transcripts are rapidly induced upon mitogenic stimulation and are downregulated in quiescent cells (35, 36). Two basic structural regions of c-Myc protein are absolutely required for its biological functions. One is the NH2-terminal transactivation domain, and the other is the COOH-terminal basic helix-loop-helix/leucine zipper domain that mediates dimerization with its cellular binding partner, Max (19). Myc/Max heterodimers bind to the DNA at the E-box sequence (CACGTG) and at other related sequences and activate transcription of c-Myc target genes (2, 5, 6). Max protein also interacts with transcriptional repressors such as Mad and Mxi1, which presumably inhibit expression of c-Myc target genes (41, 51). The activated Myc/Max heterodimers have multiple diverse activities including potentiation of cell cycle progression, inhibition of terminal differentiation, and induction of programmed cell death (2, 35, 36, 41). Constitutive expression of the c-myc gene prevents exit from the cell cycle as well as differentiation and induces apoptosis in the absence of survival cytokines (1, 4, 16). Moreover, c-myc activity is sufficient to drive resting cells into the cell cycle (4, 6), and decreased expression of c-myc gene prevents the transition from the G1 to the S phase in the cell cycle in a variety of cell types (10, 16, 19). Consistently, homozygous inactivation of c-myc in rat fibroblasts causes a marked prolongation of cell doubling time (36). However, the exact biological function of c-Myc in the regulation of normal intestinal epithelial cell growth still remains unclear.

The epithelium of mammalian intestinal mucosa is one of the most rapidly proliferating tissues in the body, being replaced every 2–3 days (23). Maintenance of mucosal integrity requires epithelial cell decisions that regulate signaling networks controlling expression of various genes involved in cell proliferation, differentiation, migration, and apoptosis (25, 33). Undifferentiated epithelial cells continuously replicate in the proliferative zone within crypts and differentiate as they migrate up the luminal surface to replace lost cells under physiological conditions. This rapid, dynamic turnover rate of the intestinal epithelium is highly regulated and critically controlled by numerous factors, including cellular polyamines (34, 37, 57, 58). The natural polyamines spermidine and spermine, and their precursor, putrescine, are organic cations found in all eukaryotic cells (37, 54). Polyamines have been implicated in a wide variety of biological functions, and regulation of cellular polyamines has been recognized to be the central convergence point for the multiple signaling pathways driving epithelial cell functions. An increasing body of evidence indicates that polyamines, either synthesized endogenously or supplied luminally, stimulate normal intestinal epithelial cell proliferation and enhance differentiation during the early stage of mucosal development and that deceased levels of cellular polyamines inhibit mucosal growth and maturation (28–30, 32, 34, 42, 57–59). To define the exact role of polyamines in cell proliferation at the molecular level, it has been shown that increased polyamines are necessary for the stimulation of c-myc gene expression in vitro (59, 60) and in vivo (56) and that polyamines regulate transcription but not posttranscription of the c-myc gene in normal intestinal epithelial cells (43).

On the basis of the fact that c-myc gene expression increases rapidly in regenerative mucosal tissues following injury (56) and that its activation requires cellular polyamines (43, 59), it is reasonable to consider the possibility that polyamine-induced expression of the c-myc gene plays an important role in the stimulation of normal intestinal epithelial cell proliferation. To test the hypothesis, we first examined whether polyamine-induced c-Myc forms heterodimers with Max and binds to the DNA at the E-box sequence in IEC-6 cells. Second, we determined whether manipulating levels of c-Myc protein, either decreased by using specific c-myc antisense oligodeoxyribonucleotides or increased by transient infection with the recombinant adenoviral vector containing wild-type c-myc cDNA, would alter intestinal epithelial cell growth in the presence or absence of cellular polyamines. Data presented here demonstrate that c-Myc is involved in the regulation of normal intestinal epithelial cell proliferation and that polyamines are required for the stimulation of mucosal growth through activation of c-myc gene expression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals and supplies. Disposable culture ware was purchased from Corning Glass Works (Corning, NY). Tissue culture media and dialyzed fetal bovine serum (dFBS) were obtained from Invitrogen (Carlsbad, CA), and biochemicals were obtained from Sigma (St. Louis, MO). The double-stranded oligonucleotides used in gel shift assay were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). c-Myc antisense or sense oligodeoxyribonucleotides were purchased from BIOGNOSTIK (Göttingen, Germany). The monoclonal antibodies against c-Myc and Max were purchased from BD Biosciences Clontech (Palo Alto, CA), and {alpha}-difluoromethylornithine (DFMO) was obtained from Ilex Oncology (San Antonio, TX). The DNA probes pSV2neo containing rat c-Myc, Max, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA were obtained from American Type Culture Collection (ATCC; Manassas, VA). [{alpha}-32P]dCTP (3,000 Ci/mmol) and [{alpha}-32P]UTP (800 Ci/mmol) were purchased from Amersham (Arlington Heights, IL), and [{alpha}-3H]thymidine (2 Ci/mmol) was obtained from New England Nuclear (Boston, MA).

Recombinant adenovirus construction and infection. Adenoviral vectors were constructed using the Adeno-X expression system (Clontech) according to the protocol provided by the manufacturer. Briefly, the cDNA of human c-myc was cloned into the pShuttle by digesting the pCMV-Myc with BamH I/HindIII and ligating the resultant fragments into the XbaI site of the pShuttle vector. pAdeno-c-myc (Admyc) was constructed by digesting pShuttle construct with PI-SceI/I-CeuI and ligating the resulting fragment into the PI-SceI/I-CeuI sites of the pAdeno-X adenoviral vector. Recombinant adenoviral plasmids were packaged into infectious adenoviral particles by transfecting human embryonic kidney (HEK)-293 cells by using Lipofectamine Plus reagent. The adenoviral particles were propagated in HEK-293 cells and purified upon cesium chloride ultracentrifugation. Titers of the adenoviral stock were determined by standard plaque assay. Recombinant adenoviruses were screened for the expression of the introduced gene by fluorescence microscopy and Western blot analysis by using the specific anti-c-Myc antibody. pAdeno-X, which was the recombinant replication-incompetent adenovirus carrying no c-myc cDNA insert (Adnull), was grown and purified as described above and served as a control adenovirus. Cells were infected by various concentrations of Admyc or Adnull, and cell samples were collected for various measurements at 24 or 48 h after the infection.

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. (48). IEC-6 cells originated from intestinal crypt cells as judged by morphological and immunologic criteria. They are nontumorigenic and retain the undifferentiated character of intestinal crypt cells. Stock cells were maintained in T-175 flasks in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% heat-inactivated FBS, 10 µg/ml insulin, and 50 µg/ml gentamicin sulfate. 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; the medium was changed three times weekly. The cells were restarted from original frozen stock every seven passages. Tests for mycoplasma were routinely negative. Cells from passages 15 to 20 were used in the experiments. There were no significant changes in biological function and characterization from passages 15 to 20.

In the first series of studies, we examined whether polyamines are required for stimulation of c-myc gene expression by exposure to 5% dFBS and whether polyamine-induced c-Myc form heterodimers with Max and have the sequence-specific DNA binding activity. The general protocol of the experiments and the methods were similar to those described previously (30, 32). Briefly, IEC-6 cells were plated at 6.25 x 104 cells/cm2 and grown in control medium (DMEM containing 5% dFBS, 10 µg/ml insulin, and 50 µg/ml gentamicin sulfate) with or without 5 mM DFMO for 4 days. Before experiments, cells were serum deprived for 24 h and then exposed to 5% dFBS. The ornithine decarboxylase (ODC) activity, cellular polyamines, c-myc and max mRNAs and proteins, sequence-specific Myc/Max DNA binding activity, and DNA synthesis were measured at different times after administration of 5% dFBS. The dishes were placed on ice, the monolayers were washed three times with ice-cold Dulbecco's phosphate-buffered saline (D-PBS), and then different solutions were added according to the assays to be conducted.

In the second series of studies, we determined whether the observed decrease in c-myc expression in response to the known stimulator 5% dFBS in polyamine-deficient cells results from inhibition of its gene transcription. Cells were grown in the presence or absence of DFMO for 4 days and then serum deprived for 24 h before experiments. The transcription rates of c-Myc were measured 3 h after exposure to 5% dFBS by performing nuclear run-on transcription assays.

In the third series of studies, we investigated whether manipulating c-Myc, either decreased using c-myc antisense oligodeoxyribonucleotides or increased by transient infection with the Admyc, alters cell growth in the presence or absence of cellular polyamines. Cells were grown for 4 days and then exposed to different concentrations of phosphothionate-modified c-myc antisense or sense oligodeoxyribonucleotides. Levels of c-Myc protein and cell proliferation were examined 48 h after treatment with the antisense or sense oligomers. In a separate experiment, ectopic expression of the wild-type c-myc gene was induced by the transient infection of normal IEC-6 cells with the Admyc, and cell proliferation was measured 24, 48, and 72 h after infection.

Assay for ODC activity. ODC activity was determined by using a radiometric technique in which the amount of 14CO2 liberated from DL-[14C]ornithine (Sigma) was estimated (50). Sample collection and the assay procedure were carried out as described in previous publications by our laboratory (57, 58). Enzymatic activity was expressed as picomoles of CO2 per milligram of protein per hour.

Polyamine analysis. The cellular polyamine content was analyzed by performing high-performance liquid chromatographic (HPLC) analysis as previously described (57, 59). Briefly, after the cells were washed three times with ice-cold D-PBS, 0.5 M perchloric acid was added, and the cells were frozen at –80°C until ready for extraction, dansylation, and HPLC analysis. The standard curve encompassed 0.31–10 µM. Values that fell >25% below the curve were considered undetectable. The results are expressed as nanomoles of polyamine per milligram of protein.

Measurements of DNA synthesis and cell number. DNA synthesis in cultured cells was measured with the use of [3H]thymidine incorporation techniques as previously described (24, 55). Cells in 24-well plates were pulsed with 1 µCi/ml [3H]thymidine for 4 h before harvest. Cells were washed twice with cold D-PBS solution and then incubated in cold 10% trichloroacetic acid (TCA) for 30 min at 4°C. After being rinsed twice with 10% TCA, cells were dissolved in 0.5 ml of 0.5 N NaOH at 37°C in humidified air for 90 min. The incorporation of [3H]thymidine into DNA was determined by counting the aliquot of cell lysate in a Beckman liquid scintillation counter. The protein content of an aliquot of cell lysate was determined by using the method described by Bradford (7). DNA synthesis was expressed as disintegrations per minute per microgram of protein. Cell numbers were counted according to the standard Trypan blue staining method as described previously by our laboratory (59).

RNA isolation and Northern blot analysis. Total RNA was extracted with the RNAeasy mini kit from Qiagen by following the instructions provided by the manufacturer. Final RNA was dissolved in water and estimated from its ultraviolet (UV) absorbance at 260 nm, using a conversion factor of 40 units. In most cases, 30 µg of total cellular RNA were denatured and fractionated electropheretically by 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 5x Denhardt's solution, 5x standard saline citrate (SSC), 50% formamide, 25 mM potassium phosphate, and 100 µg/ml denatured salmon sperm DNA. The cDNA probes for c-myc, max, 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 1x SSC-0.1% (SDS) for 10 min at room temperature. After the final wash, the filters were autoradiographed with intensifying screens at –70°C.

Nuclear run-on transcription assays. Nuclei were prepared according to the established methods of deBustros et al. (14). Briefly, IEC-6 cells were suspended in buffer A (20 mM Tris·HCl, pH 7.4, 10 mM NaCl, and 3 mM MgCl2). Nonidet P-40 was added at a final concentration of 0.1%, and the suspension was homogenized in a sterile Dounce homogenizer. Nuclei were pellet 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/ml DNA 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 (39). 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) at 26°C for 10 min. The RNA was extracted with the RNAeasy mini kit. The cDNAs for c-myc (BamH I), GAPDH (HindIII), and pSV2neo were linearized by digestion with the respective enzymes and were boiled and blotted (20 µg of cDNA per blot) onto nitrocellulose membrane (Gene Screen; DuPont). 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 2x SSC-1% SDS at 65°C for 1 h and then in 0.1x SSC-0.1% SDS at room temperature for 1 h. Filters were exposed to Kodak XAR-2 film at –70°C.

Western blot analysis. Cell samples, placed in SDS sample buffer (250 mM Tris·HCl, pH 6.8, 2% SDS, 20% glycerol, and 5% mercaptoethanol), were sonicated and then centrifuged (12,000 rpm) at 4°C for 15 min. The supernatant from cell samples was boiled for 5 min and then subjected to electrophoresis on 7.5% acrylamide gels according to the method of Laemmli (26). After the transfer of protein onto nitrocellulose filters, the filters were incubated for 1 h in 5% nonfat dry milk in 1x phosphate-buffered saline containing Tween 20 [PBS-T: 15 mM NaH2PO4, 80 mM Na2HPO4, 1.5 M NaCl, pH 7.5, and 0.5% (vol/vol) Tween 20]. Immunologic evaluation was then performed for 1 h in 1% BSA/PBS-T buffer containing 1 µg/ml of specific antibody against c-Myc or Max proteins. The filters were subsequently washed with 1x PBS-T and incubated for 1 h with the second antibody conjugated to peroxidase by protein cross-linking with 0.2% glutaraldehyde. After extensive washing with 1x PBS-T, the immunocomplexes on the filters were reacted for 1 min with chemiluminescence reagent (NEL-100; DuPont NEN). Finally, the filters were placed in a plastic sheet protector and exposed to autoradiography film for 30 or 60 s.

Preparation of nuclear protein and electrophoretic mobility shift assays. Nuclear proteins were prepared according to a procedure described previously (61), and the protein contents in nuclear preparation were determined by using the Bradford method (7). The double-stranded oligonucleotides used in these experiments included 5'-GGAAGCAGACCACGTGGT CTGCTTCC-3', which contains a consensus Myc/Max binding site (underlined). The oligonucleotides were radioactively end-labeled with [{gamma}-32P]ATP and T4 polynucleotide kinase (Promega, Madison, WI). For mobility shift assays, 0.035 pmol of 32P-labeled oligonucleotides (~30,000 cpm) and 10 µg of nuclear protein were incubated in a total volume of 25 µl in the presence of 10 mM Tris·HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 5% glycerol, and 1 µg of poly(dI-dC). The binding reactions were allowed to proceed at room temperature for 20 min. Thereafter, 2 µl of bromphenol blue (0.1% in water) were added, and protein-DNA complexes were resolved by electrophoresis on nondenaturing 5% polyacrylamide gels and visualized by autoradiography. The specificity of binding interactions was assessed by competition with an excess of unlabeled double-stranded oligonucleotide of identical sequence.

Statistical analysis. All data are expressed as means ± SE from six dishes. Autoradiographic and immunohistochemical staining results were repeated three times. The significance of the difference between means was determined by analysis of variance. The level of significance was determined using Duncan's multiple-range test (20).


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
c-myc gene expression in response to mitogenic stimulation in normal and polyamine-deficient cells. To determine the role of cellular polyamines in the stimulation of c-myc gene expression in normal intestinal epithelial cells, we chose 5% dFBS (one of the most powerful mitogens) as the c-myc stimulator. Cells were grown in the presence or absence of 5 mM DFMO for 4 days and then exposed to 5% dFBS after 24-h serum deprivation. Treatment with 5% dFBS increased ODC activity and cellular polyamine levels in normal quiescent IEC-6 cells (without DFMO). As shown in Fig. 1A, ODC activity increased 1 h after 5% dFBS was added, peaked at 2–4 h after addition, and then began to decrease. Although a significant decrease in ODC activity occurred at 12 h, it remained higher than the control value at 24 h thereafter. Increased ODC activity correlated well with an increase in the levels of the cellular polyamines putrescine, spermidine, and spermine after exposure to 5% dFBS (Fig. 1B). On the other hand, ODC activity in DFMO-treated cells was inhibited totally (Fig. 1A) and basal cellular polyamine levels were decreased dramatically (Fig. 1B). Exposure to 5% dFBS after 24-h serum deprivation caused no additional increase in ODC activity and cellular polyamine levels in the presence of DFMO. In fact, the levels of putrescine and spermidine were undetectable, and spermine content was decreased by ~60% in DFMO-treated cells regardless of the presence or absence of 5% dFBS. Although the exact reasons why cellular spermine was not completely depleted are unclear, the ~40% of intracellular spermine remaining in DFMO-treated cells did not result from the culture medium containing dFBS without polyamines.



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Fig. 1. Serum-induced ornithine decarboxylase (ODC) activity and polyamine levels in control and {alpha}-difluoromethylornithine (DFMO)-treated quiescent IEC-6 cells. A: ODC activity. Cells were grown in the presence or absence of 5 mM DFMO for 4 days and serum deprived for 24 h before experiments. ODC activity was measured at different times after addition of 5% dialyzed serum (dFBS). Values are means ± SE from 6 dishes. *P < 0.05 compared with groups at 0 h postserum. B: cellular polyamine concentrations in cells described in A. Polyamine levels were measured using high-performance liquid chromatographic analysis 4 h after exposure to 5% dFBS. Values are means ± SE from 6 dishes. *P < 0.05 compared with groups at 0 h postserum. +P < 0.05 compared with controls.

 
Induced cellular polyamines are essential for the stimulation of c-myc gene expression in IEC-6 cells. Increased polyamine levels resulting from exposure to 5% dFBS were paralleled by an activation of c-myc mRNA expression in control cells (Fig. 2A). Levels of c-myc mRNA were increased by approximately six times the control value between 2 and 6 h after the treatment. In contrast, basal levels of c-myc mRNA in polyamine-deficient cells were lower than those observed in control cells, and there was little change in c-myc mRNA level after exposure to 5% dFBS (Fig. 2B). In addition, treatment with 5% dFBS failed to alter expression of max mRNA in IEC-6 cells. There were no significant changes in levels of max mRNA in either control or polyamine-deficient cells after exposure to 5% dFBS.



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Fig. 2. Changes in expression of c-myc and max mRNA in IEC-6 cells described in Fig. 1. A: representative autoradiograms of Northern blots for c-myc and max mRNA in control cells (without DFMO pretreatment). Total cellular RNA (30 µg) was isolated and examined by Northern blot analysis using a 32P-labeled c-myc or max cDNA probe. Hybridization to a labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe served as a marker for equal loading of lanes. B: representative autoradiograms of Northern blots in DFMO-treated cells. Three experiments were performed that showed consistent results.

 
Changes in sequence-specific c-myc DNA binding activity following increases in cellular polyamine levels. Induced levels of c-myc mRNA were paralleled by an increase in nuclear c-Myc protein in quiescent control cells exposed to 5% dFBS (Fig. 3 Aa). Furthermore, increased levels of nuclear c-Myc protein were accompanied by increases in the sequence-specific DNA binding activity (Fig. 3Ab). The marginal increase in c-Myc binding activity was observed at 1 h after administration of 5% dFBS, and the maximum increase occurred at 6 h thereafter. In contrast, there was no significant change in the Max protein level after treatment with 5% dFBS. Because changes in c-Myc protein level correlated well with the induced DNA binding activity in cells exposed to 5% dFBS, increases in Myc/Max heterodimers resulted primarily from increased nuclear c-Myc protein, based on examination of the ratio of c-Myc to Max. Depletion of cellular polyamines by DFMO prevented the increases in nuclear c-Myc protein and in its binding activity induced by 5% dFBS (Fig. 3B). In polyamine-deficient cells, basal levels of nuclear c-Myc protein and Myc DNA binding activity were actually significantly lower than in controls. Administration of 5% dFBS slightly increased Myc binding activity in the absence of cellular polyamines, although there were no significant changes in the levels of nuclear c-Myc and Max proteins. These results indicate that increasing cellular polyamine levels by exposure to 5% dFBS induces nuclear c-Myc protein that has specific DNA binding activity in normal intestinal epithelial cells.



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Fig. 3. Changes in levels of nuclear c-Myc and Max proteins and their sequence-specific DNA binding activity in IEC-6 cells described in Fig. 1. A: representative autoradiograms of Western immunoblots (a) and Myc/Max binding activity (b) in control cells. Nuclear extracts were prepared at different times after exposure to 5% dFBS, and levels of c-Myc and Max proteins were examined using Western blot analysis. The sequence-specific DNA binding activity of Myc/Max in the nuclear extracts compared with those used for Western blot analysis was measured by performing electrophoretic mobility shift assays using 10 µg of nuclear protein and 0.035 pmol of 32P-end-labeled oligonucleotides containing a consensus c-Myc binding site. Positions of the specifically bound DNA-protein complexes are indicated. B: representative autoradiograms of Western immunoblots (a) and Myc/Max binding activity (b) in DFMO-treated cells. Three experiments were performed that showed similar results.

 
To evaluate the specificity of the DNA binding reactions shown in Fig. 3, we performed competitive inhibition experiments. As shown in Fig. 4A, Myc binding activity in control and polyamine-deficient cells was dose-dependently inhibited when various concentrations of the unlabeled oligonucleotide containing a single Myc/Max binding site were added to the binding reaction mixture. We also examined the effects of the unlabeled oligonucleotide containing a mutated Myc/Max binding site on the binding activity and demonstrated that the mutated oligonucleotide did not inhibit Myc binding activity (data not shown). To examine the compositions of induced binding complexes that occur after administration of 5% dFBS in normal quiescent cells, we determined effects of anti-c-Myc and anti-Max antibodies added to the binding reaction mixture on Myc binding activity. It was expected that binding of either anti-c-Myc or anti-Max antibody would inhibit the specific DNA binding activity, thereby preventing the expected supershift in this study. Results presented in Fig. 4B clearly show that anti-c-Myc or anti-Max antibody, when preincubated with the nuclear extract from normal quiescent cells exposed to 5% dFBS, almost completely inhibited the Myc/Max binding activity. In contrast, preincubation with the control (anti-c-Jun) antibody had no inhibitory effect on c-Myc binding activity (data not shown). These results indicate that induced c-Myc binding activity is specific to the Myc/Max binding site and is mediated through the c-Myc/Max heterodimers.



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Fig. 4. Effects of addition of unlabeled Myc/Max consensus oligonucleotide or antibody against c-Myc or Max to the binding reaction mixture on the sequence-specific Myc/Max binding activity in IEC-6 cells. A: representative autoradiograms of competitive inhibition experiments: controls (a) and DFMO-treated cells (b). Studies were performed by incubating 10 µg of nuclear protein with 0.035 pmol of 32P-end-labeled Myc/Max consensus oligonucleotide in the presence of different concentrations of unlabeled Myc/Max consensus oligonucleotides. Positions of the specifically bound DNA-protein complex and the freely migrating probes are indicated. B: effects of addition of the antibody against c-Myc (a) or Max (b) on the Myc/Max binding activity. Nuclear extracts (10 µg) were initially incubated with 1 or 2 µg of the indicated antibody for 40 min and then subjected to electrophoretic mobility shift assays as described in A. Three experiments were performed that showed consistent results.

 
Effect of polyamines on c-myc gene transcription. This experiment was performed to determine the possibility that polyamines were involved in the stimulation of c-myc gene expression at the transcription level. As shown in Fig. 5A, depletion of cellular polyamines by DFMO resulted in a decrease in the basal transcription of the c-myc gene in IEC-6 cells. The rate of c-myc gene transcription in DFMO-treated cells was decreased by ~65%, which was completely prevented by exogenous spermidine given together with DFMO. On the other hand, transcription rate of the c-myc gene was significantly stimulated by 5% dFBS in normal quiescent cells, and the induced rate of c-myc gene transcription was ~2.8 times the control level (Fig. 5B). However, polyamine depletion prevented the increased transcription of the c-myc gene in the DFMO-treated cells exposed to 5% dFBS. The transcription rate of the c-myc gene in polyamine-deficient cells increased slightly after administration of 5% dFBS and was ~70% of the control level. These results indicate that polyamines are necessary for the stimulation of c-myc gene transcription in normal intestinal epithelial cells.



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Fig. 5. Rate of c-myc gene transcription in response to 5% dFBS in the presence or absence of cellular polyamines. A: alterations in the rate of c-myc gene transcription after polyamine depletion. a: Representative autoradiograms of nuclear run-on transcription assays. Cells were grown in control cultures and cultures containing either 5 mM DFMO or DFMO plus 5 µM spermidine (SPD) for 4 days. Nuclei were isolated, and the rate of transcription of the c-myc gene was measured by nuclear run-on transcription analysis. Conditions for nuclear run-on assay are described in MATERIALS AND METHODS. Equal amounts of [32P]UTP-labeled RNA (5x106 cpm) were hybridized to the filter containing immobilized plasmids of c-myc, GAPDH, and control pSV2neo. b: Quantitative analysis derived from densitometric scans of nuclear run-on transcription analysis of c-myc from cells described in Aa. Relative transcription rates were normalized as measured by densitometry of GAPDH, and values are means ± SE of data from 3 separate experiments. *P < 0.05 compared with controls and DFMO+SPD. B: serum-induced c-myc gene transcription in control and DFMO-treated quiescent cells. a: Representative autoradiograms of nuclear run-on transcription assays. Cells were grown in the presence or absence of DFMO for 4 days and then serum deprived for 24 h before experiments. Rate of c-myc gene transcription was measured 4 h after exposure to 5% dialyzed serum (dFBS). b: Quantitative analysis derived from densitometric scans of nuclear run-on transcription analysis of c-myc from cells described in Ba. Values are means ± SE of data from 3 separate experiments. *P < 0.05 compared with cells exposed to no dFBS. +P < 0.05 compared with control cells exposed to 5% dFBS.

 
Association of activation of c-myc gene expression with an increase in DNA synthesis. In normal quiescent IEC-6 cells (without DFMO), exposure to 5% dFBS not only increased c-myc gene expression and sequence-specific Myc/Max binding activity (Fig. 6A) but also stimulated cell proliferation, as indicated by an increase in DNA synthesis (Fig. 6B). Increases in c-myc gene expression and Myc/Max binding activity preceded an induced rate of DNA synthesis after exposure to 5% dFBS. The maximum increases in the binding activity occurred at 6 h, whereas DNA synthesis increased significantly 12 h after administration of 5% dFBS. Consistent with the inhibitory effect of polyamine depletion on c-myc gene expression and Myc/Max binding activity, the DNA synthesis in polyamine-deficient cells just slightly increased after treatment with 5% dFBS. These findings suggest that polyamine-modulated c-myc expression and resultant formation of Myc/Max binding complexes play an important role in the process by which cell proliferation is stimulated by 5% dFBS.



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Fig. 6. Association of induced Myc binding activity and cellular DNA synthesis in control and DFMO-treated quiescent IEC-6 cells after exposure to 5% dFBS. A: relative levels of the sequence-specific Myc/Max binding activity as measured by quantitative analysis of gel shift results from cells described in Fig. 3. Cells were grown in the presence or absence of 5 mM DFMO for 4 days and then serum deprived for 24 h before experiments. Nuclear extracts were isolated at different times after exposure to 5% dFBS, and Myc/Max binding activity was measured by electrophoretic mobility shift assays. Values are means ± SE of data from 3 separate experiments. *P < 0.05 compared with 0 h postserum groups. +P < 0.05 compared with control cells exposed to 5% dFBS. B: cellular DNA synthesis in cells described in A. DNA synthesis was measured using the [3H]thymidine incorporation technique (DPM, disintegrations per minute). Values are means ± SE from 6 cultures. *P < 0.05 compared with 0 h postserum groups. +P < 0.05 compared with control cells exposed to 5% dFBS.

 
Effect of inhibition of c-myc gene expression by the specific antisense oligodeoxyribonucleotide on cell growth. To elucidate the exact role of polyamine-modulated c-myc expression in normal intestinal epithelial cell proliferation, we carried out a series of experiments using the specific c-myc antisense oligomers in IEC-6 cells. In the first study, we examined cellular uptake of c-myc antisense oligomers by using the FITC-labeled oligodeoxyribonucleotide. The significant accumulation of c-myc antisense oligomers occurred at 2 h and was linearly increased for up to 12 h after the administration. The fluorescence staining for c-myc antisense oligomers was visible under microscopy and present inside the cytoplasm (data not shown), indicating that these c-myc antisense oligomers are easily taken up by IEC-6 cells.

In the second study, we examined whether decreasing c-Myc protein levels by adding the c-myc antisense oligomers altered the cell proliferation in normal IEC-6 cells (without DFMO). Cells were grown in the standard medium for 48 h and then exposed to different concentrations of c-myc antisense oligomers. Control cells were treated with the matched randomized control oligomers at the same concentrations. DNA synthesis and cell number were measured 48 h after treatment with c-myc antisense or control oligomers. Results presented in Fig. 7, A and B, show that administration of c-myc antisense oligomers significantly inhibited the rate of DNA synthesis and final cell numbers. When various doses of c-myc antisense oligomers were tested, the dose of 2 µM tended to decrease cell growth, but the difference was not statistically significant. At the concentration of 4 µM, however, the DNA synthesis and cell numbers were markedly inhibited. The control oligomers at the same concentrations showed no inhibitory effects. Figure 7C further shows that c-myc antisense oligomers dramatically inhibited expression of c-Myc protein, whereas control oligomers at the same concentration did not.



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Fig. 7. Effect of inhibition of c-Myc expression by the specific antisense oligomers on cell proliferation in normal IEC-6 cells. A: DNA synthesis. Cells were grown in standard DMEM medium (without DFMO) for 48 h and exposed to the c-myc antisense oligomers. Control cells were treated with the matched randomized control oligomers. DNA synthesis was measured 48 h after administration of the antisense or control oligomers by using the [3H]thymidine incorporation technique. Values are means ± SE from 6 cultures. *P < 0.05 compared with controls. B: cell numbers in groups of cells described in A. C: representative autoradiograms of Western immunoblots. Whole cell lysates were harvested, applied to each lane (20 µg) equally, and subjected to electrophoresis and immunoblotting. After the blot was stripped, actin immunoblotting was performed as an internal control for equal loading. Three experiments were performed that showed similar results. Con, control.

 
In the third study, we examined the effect of c-myc antisense oligomers on cell proliferation in cells treated with DFMO plus spermidine. As shown in Fig. 8, c-myc gene expression and cell proliferation decreased when cells were grown in the presence of DFMO for 6 days. Spermidine added concomitantly with DFMO was able to maintain the gene expression and growth at control levels. Exposure to c-myc antisense oligomers at the concentration of 4 µM for 48 h not only decreased c-Myc protein levels but also prevented restoration of cell growth by spermidine in DFMO-treated cells. These results indicate that c-Myc is involved in the regulation of normal intestinal epithelial cell proliferation and that increased expression of the c-myc gene is essential for the stimulation of cell proliferation by polyamines.



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Fig. 8. Effect of c-myc antisense oligomers on restoration of cell growth and c-Myc expression by exogenous SPD in polyamine-deficient cells. A: cell numbers. After cells were grown in DMEM containing 5 mM DFMO with or without 5 µM SPD for 4 days, the specific c-myc antisense (AS) or control (Con) oligomers were added to the medium. Cell numbers were measured 48 h after administration of the oligomers. Values are means ± SE from 6 cultures. *P < 0.05 compared with untreated controls and cells treated with DFMO + SPD. +P < 0.05 compared with cells treated with control oligomers. B: representative autoradiograms of Western immunoblots for c-Myc protein. Levels of c-Myc protein were measured by Western blot analysis, and equal loading was monitored by immunoblotting of actin. Three experiments were performed that showed similar results.

 
Effect of overexpression of c-myc gene on normal IEC-6 cell growth. To further define the function of c-Myc in normal intestinal epithelial cell proliferation, we examined the effect of overexpression of the c-myc gene on IEC-6 cell growth. The adenoviral vector containing the corresponding c-myc cDNA under the control of the human cytomegalovirus immediate-early gene promoter was constructed (Fig. 9A) and used in this study. The reason we chose adenoviral vectors over other methods of transfection is that adenoviral vectors have been shown to infect a variety of cultured rat and human epithelial cells with nearly 100% efficiency (32, 49). We have demonstrated that >95% of IEC-6 cells were positive when they were infected for 24 h with the adenoviral vector encoding green fluorescent protein (GFP) as the marker. Consistently, the c-Myc protein was expressed in amounts increasing with the viral load and was ~30 times the control level when adenovirus at the concentration of 100 plaque-forming units/cell was used (Fig. 9Ac). An adenovirus that lacked exogenous c-myc cDNA (Adnull) was used as negative control in this experiment and did not induce c-Myc levels. Overexpression of the c-myc gene significantly increased IEC-6 cell proliferation (Fig. 9B). Cell numbers were increased by ~12% at 24 h and by ~17% at 48 and 72 h after the infection, respectively. These results indicate that increased c-Myc expression plays a critical role in the stimulation of gut mucosal epithelial cell proliferation.



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Fig. 9. Effect of overexpression of c-myc gene on cell growth in IEC-6 cells. A: ectopic expression of the c-myc gene. a: Structure of the adenoviral vectors. The complete open reading frame of the human c-myc cDNA was amplified by PCR, sequenced, and then cloned to pShuttle of the Adeno-X expression system. PCMV, human cytomegalovirus immediate-early promoter. b: Representative autoradiograms of Western blots for c-Myc protein. Cells were infected with the Admyc or Adnull at a multiplicity of infection of 10–100 plaque-forming units (pfu)/cell, and expression of c-Myc protein was analyzed 24 h after the infection. c: Quantitative analysis of Western blots by densitometry from results described in b. Values are means ± SE of data from 3 separate experiments, and relative levels of c-Myc protein were corrected for protein loading as measured by densitometry of actin. *P < 0.05 compared with cells infected with the Adnull vector lacking c-Myc cDNA. B: cell growth. After cells were grown in standard DMEM for 24 h, they were infected with either the Admyc or Adnull vector. Assays were performed at various times after infection. Values are means ± SE of data from 3 dishes. *P < 0.05 compared with cells infected with Adnull.

 

    DISCUSSION
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Our previous studies demonstrated that regenerative gastric and duodenal mucosa after stress-induced damage exhibits induced expression of the c-myc gene following increased polyamine levels (56). Depletion of cellular polyamines inhibits c-myc expression, which is associated with a decrease in cell renewal and a delay in mucosal healing in rats. In in vitro studies, we confirmed these in vivo findings by demonstrating that polyamines are necessary for c-myc gene expression and that polyamines are involved in transcription of c-myc but have no effect on its posttranscription (43, 59). In the present studies, we have provided new evidence that polyamine-induced expression of the c-myc gene plays a critical role in the stimulation of normal intestinal epithelial cell proliferation. Induced c-Myc protein following increased polyamine levels formed heterodimers with its binding partner, Max, and specifically bound to the DNA at the E-box sequence (Fig. 3). Disruption of c-Myc expression by using specific c-myc antisense oligomers not only inhibited normal cell proliferation (without DFMO) but also prevented the restoration of cell growth by exogenous spermidine in polyamine-deficient cells (Figs. 7 and 8). Furthermore, ectopic expression of the c-myc gene by transient infection with the recombinant adenoviral vector containing wild-type c-myc cDNA significantly increased normal intestinal epithelial cell proliferation (Fig. 9).

The findings reported here clearly indicate that cellular polyamines regulate expression of c-myc but not max in normal intestinal epithelial cells. Induction of c-myc expression by treatment with 5% dFBS was almost completely prevented in polyamine-deficient cells, whereas levels of max mRNA were not altered, regardless of the presence or absence of cellular polyamines. Results presented in Fig. 5 further show that polyamines are required for stimulation of c-myc gene expression primarily through the upregulation of its mRNA synthesis, because depletion of cellular polyamines prevented the induced rate of c-myc gene transcription in cells exposed to 5% dFBS. These results confirm our previous findings (43, 56) and are consistent with those of others (8, 9) who demonstrated that polyamines stimulate transcription of c-myc and c-jun genes in COLO 320 human colon carcinoma cells. On the other hand, it is not surprising that expression of the max gene is insensitive to changes in levels of cellular polyamines in IEC-6 cells, because max is not a member of the class of mitogen-inducible genes and its expression is independent of the state of cell proliferation (19). In fact, Max levels remain relatively constant under all growth conditions (5, 6), and its mRNA is very stable (27, 46).

Induced c-Myc protein following increased polyamine levels forms heterodimers with Max, which is capable of recognizing the E-box sequence CACGTG in normal intestinal epithelial cells. There was still a slight increase in c-Myc/Max binding activity in polyamine-deficient cells exposed to 5% dFBS (Fig. 3Bb). Although the exact mechanism involved in this process is unclear, this minor stimulation could be mediated by other factors that are independent of cellular polyamines. Myc proteins belong to the family of basic helix-loop-helix/leucine zipper transcription factors whose known biological functions are dependent on dimerization with Max (5, 6, 19, 36). Although Max homodimers lack transcriptional activities in general, recent evidence suggests that the transcriptional activities of Myc/Max heterodimers result primarily from their ability to recruit chromatin-modifying complexes to DNA (3, 15). For example, Myc associates with the TRRAP (transformation-transactivation domain-associated protein) coactivator, which in turn binds the histone acetyltransferase GCN5, permitting Myc/Max dimers to direct acetylation of histones at E-box binding sites (17, 38). However, it is unclear at present whether specific factors, either coactivators or corepressors that associate with induced c-Myc/Max dimers following increased cellular polyamines, are recruited to form chromatin-modifying complexes in IEC-6 cells. In addition, induced Myc also can function through an E-box-independent pathway. It was recently shown (52) that c-Myc inhibits the function of transcription factor Miz-1 through direct interaction with Miz-1 protein, leading to E-box-independent repression of Miz-1-activated targets such as p15 and other cyclin-dependent kinase (CDK) inhibitors.

The results reported here also indicate that induced c-Myc following increased cellular polyamine levels plays a critical role in the stimulation of normal intestinal epithelial cell growth. The increase in c-Myc activity precedes the initiation of DNA synthesis after exposure to 5% dFBS, and inhibition of c-myc expression by polyamine depletion prevented the stimulation of DNA synthesis. Furthermore, decreased levels of c-Myc resulting from exposure to specific c-myc antisense oligomers inhibited cell growth, whereas increased levels of c-Myc caused by infection with the recombinant adenoviral vector containing c-myc cDNA induced cell proliferation. These findings are consistent with others showing that c-Myc is an essential factor that is required for activation of CDK complexes and mediates the transition from G1 to S phase during the cell cycle (11, 36, 44). Expression of the c-myc gene is induced in response to a large number of growth factors and may serve to integrate these external signals to sustain cell proliferation. Targeted deletion of either c-myc or N-myc leads to early embryonic lethality in mice (12, 53), and myc gene function is also essential for hematopoiesis and organogenesis (13). Deregulated expression of myc genes results in malignant transformation and genetic instability. In fact, amplification and mutation of the c-myc gene has been observed in ~15% of all human tumors (21). Because integrity of intestinal epithelium depends on the continuous epithelial cell renewal and these cells highly express c-myc, these findings suggest that c-myc is a biological regulator for normal intestinal mucosal growth under physiological conditions.

Despite many advances and identification of a number of c-myc target genes, the direct mediators of c-myc's effects on the cell cycle are still obscure. It has been shown that G1- to S-phase transition in the cell cycle is primarily controlled by specific activation and subsequent inactivation of CDK complexes, including cyclin D/CDK4 and cyclin E/CDK2 (5, 16). CDK4 and CDK2 are the rate-limiting steps for G1- to S-phase progression in higher eukaryotic cells, and activation of CDKs is able to shorten the G1 interval (22, 45). Recently, CDK4 and p21 were identified as target genes of c-Myc, and the increased c-myc stimulates expression of the CDK4 gene (22) but represses the p21 promoter (40). Induced c-Myc results in a rapid increase in CDK4 mRNA levels through four highly conserved c-Myc binding sites within the CDK4 promoter. Cell cycle progression is delayed in c-myc-deficient RAT1 cells, and this delay is associated with a defect in CDK4 induction. On the other hand, c-Myc downregulates p21 transcription through sequestration of Sp1, and repression of the p21 expression also contribute to the ability of c-Myc to promote cell proliferation (18, 40). It is speculated that induced c-Myc following increased polyamine levels stimulates normal intestinal epithelial cell proliferation through both activation of CDK4 and inactivation of p21. To support this possibility, we recently demonstrated that decreases in c-Myc levels resulting from polyamine depletion are associated with not only a decrease in CDK4 expression but also an increase in p21 levels (29, 59).

In summary, these results indicate that polyamines are absolutely required for activation of c-myc gene but have no effect on expression of the max gene in normal intestinal epithelial cells. Induced c-Myc protein following an increase in polyamine level forms c-Myc/Max complexes and binds to the specific DNA sequence after exposure to the mitogen factor. Polyamine-induced c-myc expression plays a critical role in the stimulation of normal intestinal epithelial cell proliferation, because decreases in c-Myc levels resulting from exposure to specific antisense oligomers inhibit cell proliferation and because ectopic expression of the c-myc gene resulting from infection with the Admyc stimulates cell growth. These findings suggest that c-Myc is a physiological regulator of normal intestinal epithelial cell proliferation and is implicated in maintenance of mucosal epithelial integrity. Polyamines are required for stimulation of intestinal epithelial cell proliferation during normal mucosal growth and healing following injury, at least partially, by virtue of their ability to enhance transcription of the c-myc gene.


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This work was supported by a Department of Veterans Affairs Merit Review Grant and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-57819, DK-61972, and DK-68491. J.-Y. Wang is a Research Career Scientist, Medical Research Service, Department of Veterans Affairs.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J.-Y. Wang, Dept. of Surgery, Baltimore Veterans Affairs Medical Center, 10 North Greene St., Baltimore, MD 21201 (E-mail: jwang{at}smail.umaryland.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* L. Liu and L. Li contributed equally to this work. Back


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