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
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ABSTRACT |
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ornithine decarboxylase; -difluoromethylornithine; growth arrest; gene expression; intestinal epithelium
The epithelium of mammalian intestinal mucosa is one of the most rapidly proliferating tissues in the body, being replaced every 23 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 (2830, 32, 34, 42, 5759). 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.
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MATERIALS AND METHODS |
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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 0.3110 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 [-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 [-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 [
-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 [
-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 [-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).
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RESULTS |
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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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DISCUSSION |
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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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GRANTS |
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FOOTNOTES |
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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.
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