Site-specific DNA methylation contributes to neurotensin/neuromedin N expression in colon cancers

Zizheng Dong1,2, Xiaofu Wang1, and B. Mark Evers1

1 Department of Surgery, The University of Texas Medical Branch, Galveston, Texas 77555; and 2 Institute of Biotechnology, Beijing 100071, People's Republic of China


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The neurotensin/neuromedin N (NT/N) gene is expressed in fetal colon, repressed in newborn and adult colon, and reexpressed in ~25% of colon cancers. Our purpose was to determine the effect of gene methylation on NT/N silencing in colon cancers. We found that the NT/N gene was expressed in human colon cancer cell line KM12C but not in KM20 colon cancer cells. Bisulfite genomic sequencing demonstrated that all CpG dinucleotides in the region from -373 to +100 of the NT/N promoter, including a CpG site in a distal consensus AP-1 site, were methylated in KM20 but unmethylated in KM12C cells. Treatment of KM20 cells with demethylating agent 5-azacytidine induced NT/N expression, suggesting a role for DNA methylation in silencing of NT/N in colon cancers. To better elucidate the mechanisms responsible for NT/N repression by DNA methylation, we performed gel shift assays using an oligonucleotide probe corresponding to the distal AP-1 consensus sequence of the NT/N promoter. Methylation of the oligonucleotide probe inhibited protein binding to the distal AP-1 site of the NT/N promoter, suggesting a potential mechanism of NT/N gene repression in colon cancers. We show that DNA methylation plays a role in NT/N gene silencing in the human colon cancer KM20 and that NT/N expression in KM12C cells is associated with demethylation of the CpG sites. DNA methylation likely contributes to NT/N gene expression noted in human colon cancers.

gene expression; colon cancer cells


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NEUROTENSIN (NT) is a tridecapeptide originally isolated from bovine hypothalami by Carraway and Leeman (9) and subsequently localized to specialized enteroendocrine cells (N cells) of the adult small bowel (reviewed in Ref. 45). NT has numerous physiological functions in the gastrointestinal tract, including stimulation of pancreatic secretion and colonic motility, inhibition of gastric and small bowel motility, and stimulation of pancreatic, small bowel, and colonic growth (1, 3, 11, 18, 19, 25, 51). Expression of the gene encoding NT and the structurally-related hexapeptide neuromedin N (designated NT/N) is developmentally regulated in the small intestine of both rats and humans in a distinctive temporal- and spatial-specific distribution (17, 20, 40). In the human colon, NT/N is expressed in the fetus during midgestation when the colon is similar to the small bowel from a morphological and functional standpoint; NT/N expression is not apparent in the colon of the newborn or the adult but is reexpressed in ~25% of colon cancers (20, 24). Previously, we have shown that constitutive (i.e., basal) NT/N expression in the endocrine cell line BON is highly dependent on the binding of transcription factors to a proximal promoter region located 50 bp upstream of the transcriptional start site (22). In contrast, Kislauskis and Dobner (33) have shown that a distal consensus activator protein (AP)-1 site is crucial for NT/N induction in the PC12 cell line, which does not constitutively express NT/N. Therefore, it appears that the induction of NT/N expression in nonexpressing cells is highly dependent on an intact distal AP-1 promoter element. In addition, it is likely that other genetic factors contribute to the complex pattern of NT/N expression noted in normal tissues and in some cancers.

DNA methylation appears to play a critical role in the expression of a number of genes during normal development and with malignant transformation (10, 16, 50). The natural substrates of the mammalian methyltransferase enzyme are the cytosines within the dinucleotide CpG (10, 16, 50). Methylation of cytosines in the CpG sequence is thought to ensure the silencing of certain tissue-specific genes in nonexpressing cells (10, 16). The precise spatial and temporal pattern of gene expression noted during development in certain tissues is thought to depend, in large part, on the gene methylation pattern, with demethylation of tissue-specific genes noted in the cell types in which they are expressed (2, 31, 50). Furthermore, alterations of DNA methylation, which can affect patterns of gene expression important for cell growth, occur in a number of human cancers, including colon cancers (35, 49). A mechanism that is increasingly being recognized as important for methylation-mediated gene silencing is the interference of transcription factor binding to the gene promoter (16, 50). Such interference has been noted for a number of known transcription factors, including AP-2, E2F, nuclear factor-kappa B, and cAMP response element binding protein (CREB)/activating transcription factor (ATF) (5, 6, 13, 27, 28). Promoter methylation may contribute to the gene expression pattern of tissue-specific genes (16, 50).

Previously, we have shown that NT/N expression is induced in the liver cancer cell line HepG2 by treatment with the demethylating agent 5-azacytidine (5-azaC) (15). Moreover, in vitro methylation of the NT/N promoter markedly inhibited NT/N promoter activity in HepG2 cells. These findings implicate DNA methylation in the expression of NT/N noted in certain ectopic tissues, such as hepatic-derived cells. However, the mechanisms contributing to NT/N repression by DNA methylation are not known. Furthermore, the role that DNA methylation plays in the expression of NT/N in other ectopic tissues, such as colon cancers, has not been determined. Therefore, the purpose of this study was twofold: 1) to determine whether methylation may play a role in NT/N gene expression in colon cancers and 2) to better elucidate the mechanisms contributing to NT/N expression with the use of a sensitive bisulfite sequencing technique, locus accessibility assays, and gel shift analyses.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Restriction, ligation, and other DNA-modifying enzymes were purchased from Promega (Madison, WI), Stratagene (LaJolla, CA), or New England Biolabs (Beverly, MA). PCR materials and Taq DNA polymerase were from Promega. Oligo(dT) cellulose (type III) was obtained from Collaborative Research (Bedford, MA), and radioactive compounds were obtained from New England Nuclear (Boston, MA). Nucleotides and poly(dI · dC) were purchased from Pharmacia LKB Biotechnology (Piscataway, NJ). Tissue culture media and reagents were obtained from GIBCO BRL (Grand Island, NY). All other reagents were of molecular biology grade and were either obtained from Sigma Chemical (St. Louis, MO) or Amresco (Solon, OH). Nitrocellulose filters were from Schleicher & Schuell (Keene, NH). Ribonuclease (RNase) protection experiments were performed using the RPA-II kit from Ambion (Austin, TX). To determine the relative size of the protection products, RNA markers (Ambion) transcribed with T7 RNA polymerase were run in parallel lanes. The constitutively expressed human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was obtained from Ambion and used to ensure integrity of the RNA samples analyzed by both Northern blot and RNase protection. The pGEM-Zf vector as well as oligonucleotides containing AP-1, glucocorticoid response element (GRE), and Sp1 consensus sites were purchased from Promega. The oligonucleotides containing c-myc and Oct-1 consensus sites were from Oncogene Science (Cambridge, MA) and Stratagene, respectively. The oligonucleotides corresponding to the wild-type sequences of NT/N promoter were synthesized by Oligos Etc. (Wilsonville, OR).

Cell culture. The human colon cancer cell lines KM20 and KM12C were obtained from Dr. Isaiah J. Fidler (M. D. Anderson, Houston, TX) (38, 39) and cultured in MEM plus 1 mmol/l sodium pyruvate, 1× solution of MEM nonessential amino acid, 2× solution of MEM nonessential vitamin mixture, and 10% fetal bovine serum (FBS). The human cell line BON, which was established in our laboratory from a pancreatic carcinoid tumor (21, 44), was cultured in DMEM and F12K at 1:1 supplemented with 5% FBS. All cells were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37°C.

RNA extraction, Northern blot, ribonuclease protection, and RT-PCR. Cells were harvested and RNA was obtained by the method of Schwab et al. (48), except that digestion with proteinase K was for 1-2 h at 50°C. Polyadenylated RNA was then selected from all samples by oligo(dT) cellulose column chromatography, and the final RNA concentration was quantified spectrophotometrically.

For Northern blot analysis, polyadenylated RNA was electrophoresed in 1.2% agarose-formaldehyde gels, transferred to nitrocellulose, and hybridized with a cRNA probe (pHNT E0.9) containing 806 bp of the human NT/N gene subcloned into the EcoR I site of a pGEM4 vector (4). Hybridization and washing conditions were as described previously (23, 38). Blots were stripped and reprobed with the constitutively expressed human GAPDH gene. RNase protection analyses were performed as described previously (23, 38) using the RPA-II kit according to the instructions of the supplier. For RT-PCR, two primers were used: P1 was 5'-GATGATGGCAGGAATGAAAATCCAG-3' (located in the first exon) and P2 was 5'-TGGTAAAAGCCCTGCTGTGACAGA-3' (located in the third exon). The RT-PCR was performed as described previously (38).

Cytosine deamination-PCR. Cytosine deamination by bisulfite treatment of single-stranded DNA and subsequent PCR amplification was performed essentially as described by Frommer et al. (26). Genomic DNA (20 µg) was first digested with EcoR I, denatured in 0.3 M NaOH for 20 min at 37°C in a volume of 100 µl, added to 1.1 ml of deamination buffer containing 2.2 M Na2S2O5 and 1 mM hydroquinone (pH 5.0), and then incubated at 55°C for 16 h. The DNA was desalted by dialyzing at 4-10°C against 2 liters of 5 mM NaAc (pH 5.2), 0.5 mM hydroquinone, 0.5 mM NaAc (pH 5.2), and H2O. Samples were concentrated in a vacuum desiccator, denatured with 0.3 M NaOH for 20 min at 37°C, neutralized with 3 M ammonium acetate (pH 7.0), and precipitated with ethanol. An aliquot of DNA was amplified with modified primers. All reactions were carried out in 100-µl volumes containing 1× PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTP (each), 20 pmol primers, and 2.5 units Taq DNA polymerase. The samples were then subjected to 35 cycles of amplification consisting of 1-min denaturation at 94°C, 45-s annealing at 58°C, and 1.5-min extension at 72°C, followed by an additional extension step at 72°C for 10 min. The amplified fragments were cloned into a pGEM5-Zf vector, and then at least 18 independent clones for each fragment were sequenced using T7 and Sp6 primers to determine the methylation pattern of individual molecules. Primers, chosen on the basis of the human genomic sequence, correspond to the following regions of the human NT/N gene (4): positions -339 to -311 bp and +102 to +130 bp for the amplification of upper strand and positions -373 to -345 bp and +134 to +158 bp for the lower strand. The modified primers were PU1 (5'-GTTTGGAGTGGGGGAATGAGAAGTG-3') and PU2 (5'-ATTGAATTTTGATTCCTACCATCATCTT-3') for the upper strand and PL1 ( 5'-GAATCTCATATTTCACAAAATCAAAATA-3') and PL2 (5'-GAAGTTAGGAGTAGTATGTATATAAGT-3') for the lower strand.

Nuclear accessibility assays. Nuclei were isolated from KM20 and KM12C and digested with EcoR I or Msp I at various concentrations. All digestions were terminated by addition of 10 mM EDTA, 0.5% SDS, and 600 µg/ml proteinase K at 55°C for 4 h. DNA was prepared by organic extraction and ethanol precipitation. DNA samples were restricted with Hind III, and fragments were separated overnight by electrophoresis through a 1% agarose gel with constant voltage (1.5-2 V/cm). After electrophoresis, the gel was soaked in 0.25 mol/l HCl for 10 min and rinsed briefly with deionized water. The DNA was denatured, neutralized, and transferred to nitrocellulose filters and then hybridized with the human NT/N DNA fragment containing the first exon, first intron, and second exon.

Electrophoretic mobility shift assays. KM20 and KM12C cell nuclear extracts were prepared according to the method of Dignam et al. (14) or Schreiber et al. (47). Synthetic oligonucleotides corresponding to the upper and lower strands of wild-type NT/N promoter sequence (-245 to -224) containing the distal consensus AP-1 site were synthesized, annealed, and labeled with [gamma -32P]ATP and T4 polynucleotide kinase. Electrophoretic mobility shift assays (EMSAs) were performed as described previously (22). The reaction mixtures contained 40,000 cpm (~4 fmol) of 32P end-labeled mock methylated or methylated oligonucleotide (methylated with Sss I methylase) and 10-20 µg nuclear protein in a final volume of 20 µl of 12.5 mM HEPES (pH 7.9), 100 mM KCl, 10% glycerol, 1 mM EDTA, 1 mM dithiothreitol, 2 µg BSA, and 3 µg poly(dI · dC) as nonspecific competitors. The reaction was incubated for 20 min at room temperature. Competition binding experiments were performed by first incubating the competitor fragment, in molar excess, with the nuclear protein and binding buffer for 10 min on ice. The labeled probe was then added, and incubation continued for 20 min at room temperature. The reaction mixtures were loaded onto 6% nondenaturing polyacrylamide gels and resolved by electrophoresis at 250 V for 2-3 h. The gels were subsequently dried and autoradiographed at -70°C.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The NT/N gene is expressed in the KM12C colon cancer line but not in KM20 cells. We have previously shown that NT/N is expressed in ~25% of colon cancers examined (24). This expression pattern appears random, with no relation to tumor site or stage. To better determine the mechanisms for NT/N expression in these "ectopic" tissues, we analyzed the two human colon cancer cell lines KM12C and KM20. We found that NT/N is expressed in KM12C cells, as noted by Northern blot (Fig. 1A). The BON cell line, which we have previously shown expresses the NT/N gene and secretes NT peptide (21, 44), was used as a positive control. In contrast, NT/N expression was not noted in the KM20 colon cancer cell line. To ensure intact RNA samples, the blot was reprobed with the constitutively expressed GAPDH gene, which was noted in all three cell lines.


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Fig. 1.   Analysis of neurotensin/neuromedin N (NT/N) expression. A: Northern blot analysis of RNA samples from KM12C and KM20 cells. Blot was probed with human NT/N probe (pHNT E0.9). KM12C demonstrated NT/N transcripts of the correct sizes (1.5 and 1.0 kb); however, NT/N expression was not apparent in KM20 cells. Blot was stripped and reprobed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to confirm intact RNA and relatively equal loading. B: RNA samples from KM12C, KM20, and BON cells hybridized with 32P-labeled human NT/N cRNA probe (pHNT E0.9) and analyzed by RNase protection. RNA from KM12C and BON demonstrate an expected protection product of 180 bases, consistent with exon 1 of the human NT/N gene; no detectable NT/N mRNA was identified in KM20. Lane 2 contains probe alone (without RNase). Lane M is the RNA molecular size marker (Ambion). C: total RNA isolated from KM12C, BON, and KM20 cells was reverse-transcribed and PCR-amplified as described in MATERIALS AND METHODS with the use of NT/N primer pairs. No Template, RT-PCR reaction performed without addition of RNA.

As confirmation of NT/N expression in KM12C cells, RNA was analyzed by both RNase protection and RT-PCR. The expected 180-base NT/N protection product, corresponding to exon 1, was noted in KM12C cells and the positive control BON but not in KM20 cells (Fig. 1B). Similarly, RT-PCR failed to detect the expression of NT/N in KM20 cells; the predicted fragment of ~380 bp was noted in both KM12C and BON (Fig. 1C). Together, these results identify NT/N expression in the KM12C colon cancer cell line but not in KM20 cells.

Analysis of the CpG sites in the human NT/N promoter. Previously, we have shown that the proximal promoter region of the NT/N gene is sufficient for basal promoter activity (22). To determine whether methylation may contribute to the NT/N expression noted in colon cancers, we evaluated the region from -373 to +100 bp of the human NT/N gene for potential CpG dinucleotides, which are natural substrates of mammalian methyltransferase (16, 50). Three CpG sites are located in this region (Fig. 2A). Interestingly, one CpG site, located at position -232, is contained within the consensus sequence of a distal AP-1 site (TGCGTCA), which is crucial for induction of the rat NT/N gene in the PC12 neuronal cell line (33). The second site (-190) is located in a CRE half-site (CGTCA), and the third site (+83) is located in the untranslated region of the first exon.


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Fig. 2.   Genomic sequencing and methylation analysis of plasmid clones derived from KM and BON cell lines after cytosine deamination-PCR. A: nucleotide sequences of the proximal human NT/N promoter. The 3 CpG dinucleotides are indicated by the asterisks (*). The distal activator protein (AP)-1 consensus site is in the black box. The TATATA box is indicated by the under- and overlines, and the ATG translational initiation codon is indicated by the underline. B: methylation analyses of clones derived from the upper strand of the proximal human NT/N promoter. Genomic DNA from KM20, KM12C, and BON cells was treated with sodium bisulfite, amplified, and cloned into the pGEM5-Zf vector. Primers (PU1 and PU2) to the NT/N upper strand were used in the PCR reaction as described in MATERIALS AND METHODS. Unmethylated Cs were converted to Ts in the KM12C and BON cell clones, whereas methylated Cs remained as Cs at sites -232 and -190 (PU1 primer) and +83 (PU2 primer) in the KM20 cell clones (arrows).

We next used a genomic sequencing method for analysis of the methylation pattern (26). Two sets of primers, each specific for the upper (PU1 and PU2) and lower (PL1 and PL2) strands of bisulfite-treated human genomic DNA, were designed for the analysis of the methylation status of the NT/N gene. The plasmid clones derived from each cell line were analyzed by DNA sequencing. This technique relies on deamination of cytosine, but not methylcytosine, to uracil by bisulfite in single-stranded DNA (12, 26, 30, 42, 52). Following PCR amplification, cloning, and conventional dideoxy sequencing, methylcytosine residues were identified as cytosines in the sequence ladder. Typical sequence profiles are shown in Fig. 2B. The analysis of plasmid clones derived from the upper strands demonstrated that all three CpG sites were methylated in KM20 cells and unmethylated in KM12C and BON cells. Analysis of the lower strands demonstrated identical results (data not shown). These findings suggest that demethylation of the CpG dinucleotides in the promoter region may be important for the expression of NT/N in certain cells. Therefore, DNA methylation may play a role in the repression of the NT/N gene in KM20 cells.

DNA methylation plays a role in NT/N gene expression. To determine the role of DNA methylation in the expression of NT/N in these colon cancer cells, KM20 cells were treated with the demethylating agent 5-azaC (29, 34) at a concentration of 8 µM for 4 days and then changed to fresh media for an additional 4 days. RNA was analyzed by RNase protection assays (Fig. 3A). Treatment of KM20 cells with 5-azaC resulted in the induction of NT/N expression. We also treated KM12C cells with 5-azaC and then determined whether NT/N expression was altered using RT-PCR; GAPDH was amplified in the same reaction vessels (Fig. 3B). As expected, 5-azaC had no apparent effect on NT/N expression. Untreated BON cells were used as a positive control for NT/N expression. Therefore, these findings suggest that demethylation of the CpG sites by 5-azaC results in NT/N gene induction in KM20 cells. Conversely, NT/N expression in KM12C is not affected by 5-azaC since the CpG sites are unmethylated.


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Fig. 3.   Effect of 5-azacytidine (5-azaC) on NT/N expression in KM20 and KM12C cells. A: RNA from untreated KM20 cells (lane 1) or KM20 cells treated with the demethylating agent 5-azaC (lane 2) analyzed by RNase protection. To confirm intact RNA, a separate RNase protection analysis was performed with the use of a human GAPDH probe (Ambion). B: total RNA isolated from untreated KM12C cells (lane 1) or KM12C treated with 5-azaC (lane 2) was reverse-transcribed and PCR-amplified using primers for NT/N and GAPDH in the same reaction vessel. BON cell RNA was used as a positive control for NT/N expression. No template, RT-PCR reaction performed without addition of RNA. M, molecular mass marker.

To confirm that treatment with 5-azaC results in demethylation of the CpG sites in the NT/N gene, we again used a genomic sequencing method to analyze DNA from KM20 cells that were either treated with 5-azaC or untreated (Fig. 4). All three CpG sites were unmethylated in KM20 cells after 5-azaC treatment. Together, these results provide further evidence that DNA methylation plays a role in the repression of the NT/N gene in the human colon cancer cell line KM20.


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Fig. 4.   Genomic sequencing and methylation analysis of KM20 cells treated with 5-azaC. Methylation analysis of the upper strand of the proximal NT/N promoter in KM20 cells is shown. Genomic DNA from KM20 cells (either treated or untreated with 5-azaC) was treated with sodium bisulfite, amplified, and cloned into the pGEM5-Zf vector. The methylated Cs (indicated by arrows) are unmethylated and converted to Ts with 5-azaC treatment.

One potential mechanism by which DNA methylation can suppress gene expression is alteration of the chromatin structure, thus influencing gene accessibility (16, 36, 50). Therefore, locus accessibility assays were performed using nuclei isolated from KM12C and KM20 cells treated with either EcoR I or Msp I restriction enzymes (37) (Fig. 5). Our results with either restriction enzyme did not reveal significant differences in the digestion profiles of either KM12C or KM20 cells, suggesting that the overall chromatin structure is similar, although the methylation status of the promoter is different in these cell lines.


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Fig. 5.   Southern blot analysis of DNA from KM cells restricted with either EcoR I or Msp I. Southern blot pattern of genomic DNA from KM20 and KM12C cell lines restricted with increasing amounts of either EcoR I (A) or Msp I (B) and probed with a labeled human NT/N probe. There was no significant difference in the digestion patterns of KM20 or KM12C treated with either of the restriction endonucleases. EcoR Ir, band resistant to EcoR I restriction; EcoR Is, bands sensitive to EcoR I restriction; Msp Ir, band resistant to Msp I restriction; Msp Is, bands sensitive to Msp I restriction.

DNA methylation inhibits the binding of transcription factors to a distal consensus AP-1 site in the NT/N promoter. We next determined whether methylation suppresses NT/N gene expression by preventing the binding of transcription factors to the promoter. To assess this possibility, an oligonucleotide corresponding to the wild-type NT/N sequence (-245 to -224) containing the distal consensus AP-1 site was synthesized and used in EMSAs with nuclear extracts from the KM12C cell line. When the human NT/N (hNT/N) promoter fragment was used as a probe, a DNA-protein binding complex was demonstrated that was effectively inhibited by unlabeled human NT/N and an oligonucleotide containing an AP-1 consensus sequence (Fig. 6A). In contrast, the complex was not affected by a molar excess of oligonucleotides containing either GRE or c-myc consensus sites. These findings demonstrate the binding of AP-1 proteins to this NT/N promoter region, as shown by the competition experiment using the AP-1 oligonucleotide probe; the absence of competition using the GRE and c-myc probes indicates that the binding is specific and is not competed by unrelated nucleotide sequences.


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Fig. 6.   Electrophoretic mobility shift assay of AP-1 protein binding to the consensus AP-1 site of the human NT/N promoter. A: nuclear extracts from KM12C cells were assessed for protein binding using an oligonucleotide probe (hNT/N -245/-224) that contained a consensus AP-1 site. Competition experiments were performed with a 100-fold molar excess of unlabeled hNT/N oligonucleotide (lane 2) as well as unlabeled AP-1, c-myc, and glucocorticoid response element (GRE) oligonucleotides (lanes 2-5). Protein binding is noted to the labeled hNT/N probe, which is effectively competed using either unlabeled hNT/N or AP-1. B: labeled hNT/N oligonucleotide probe was either methylated (M+) or unmethylated (M-) and analyzed by mobility shift assays with the use of KM12C protein extracts. Methylation of the labeled probe inhibits protein binding to the AP-1 site. C: As a control for the specificity of the gel shift results, labeled oligonucleotides containing consensus Sp1 or Oct-1 sites were either methylated or unmethylated and analyzed by mobility shift assays using KM12C protein extracts. No significant effect on the binding pattern was noted with methylation of either the Sp1 or Oct-1 oligonucleotides. Competition experiments were performed using a 100-fold molar excess of unlabeled oligonucleotide.

To further assess the role of methylation in protein binding to the NT/N promoter fragment, the NT/N oligonucleotide was methylated by Sss I methylase and labeled, and the EMSA was repeated using KM12C nuclear extracts (Fig. 6B). Methylation of the NT/N probe effectively prevented the binding of AP-1 proteins compared with the specific binding noted with the unmethylated probe. As a control for the specificity of the methylation results using the NT/N promoter fragment, nonspecific oligonucleotides containing either consensus Sp1 or Oct-1 sites were methylated and labeled, and the EMSA was repeated with KM12C extracts. Methylation of these probes did not alter the binding pattern of Sp1 or Oct-1 proteins to their consensus binding sites. Protein binding, however, was effectively competed using the unlabeled probe in molar excess. Together, these findings suggest that DNA methylation effectively blocks protein binding to this distal AP-1 promoter sequence and may explain the gene silencing noted in the KM20 cell line in which this CpG site is methylated.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we demonstrated that the terminally differentiated endocrine gene NT/N is constitutively expressed in the human colon cancer cell line KM12C but not in KM20 cells. Consistent with these results, we have previously shown that NT/N is expressed in the fetal colon, repressed in the newborn and adult colon, and then reexpressed in ~25% of human colorectal cancers (8, 24). The mechanisms underlying this pattern of NT/N gene silencing in normal adult tissues with selective reexpression in a minority of colorectal cancers have not been determined. Delineating the cellular mechanisms contributing to the complex pattern of NT/N expression will provide a better understanding of factors regulating differentiated gene expression associated with normal intestinal development and malignant transformation.

Previously, we have shown that NT/N expression is induced in the HepG2 hepatocellular cancer by treatment with the potent demethylating agent 5-azaC (15). Moreover, in vitro methylation of the NT/N promoter markedly inhibited its activity in the HepG2 cell line, thus further supporting a role for gene methylation in the expression of NT/N in certain ectopic tissues. Therefore, our present study was performed to determine whether similar mechanisms may explain the selective expression of NT/N in only certain colorectal cancers. We found that KM20 cells, in contrast to the KM12C cell line, do not express the NT/N gene as assessed by Northern blot and sensitive RNase protection and RT-PCR analyses. However, treatment of KM20 cells with 5-azaC resulted in NT/N gene activation, suggesting that gene methylation may be responsible for the NT/N gene silencing noted in KM20 cells. Therefore, these results provide direct evidence that the repressed NT/N gene can be reactivated by the inhibition of DNA methylation.

Genes with tissue-specific expression patterns often have methylated CpGs within the promoter region (16, 31, 50, 52). We next performed nucleotide sequence analysis of the NT/N proximal promoter sequences and demonstrated three CpG dinucleotides in the region from -373 to +100, which has previously been shown to be sufficient for both basal and induced NT/N expression (22, 33). As a further assessment of the role of DNA methylation in NT/N expression, we performed genomic sequencing after bisulfite treatment, which results in deamination of unmethylated cytosines, identified as thymines, but with no change in the methylated cytosines (12, 30, 42, 52). Analysis of DNA methylation by bisulfite sequencing allows the detection of methylation at a greater number of cytosines and with higher resolution than analysis with methylation-sensitive enzymes (52). Moreover, this technique has been used to assess methylation patterns in other tissue-specific genes, such as tyrosine hydroxylase, galectin-1, prolactin, and growth hormone (42, 43, 46). We found that all three of the CpG doublets in the region from -373 to +100 are methylated in KM20 cells and demethylated in KM12C and BON cells. Treatment of KM20 cells with 5-azaC resulted in demethylation of the CpG doublets and NT/N gene activation. Together, these findings demonstrating methylation of CpG doublets in the NT/N-nonexpressing KM20 cell line and CpG demethylation in the NT/N-expressing cell lines KM12C and BON further support a role for methylation in NT/N gene silencing in certain cells.

Interestingly, one of the CpG sites methylated in the KM20 cell line is contained within an AP-1 consensus site that is critical for induction of the rat NT/N gene in response to various environmental influences. Kislauskis and Dobner (33) noted that this site served as a functional focal point for NT/N gene induction in PC12 rat neuronal cells. Since one of the mechanisms by which methylation can suppress gene expression is by directly preventing the binding of transcription factors (5, 6, 13, 16, 27, 28, 50), we next determined whether DNA methylation alters protein binding to this distal AP-1 site. Methylation of the human NT/N promoter fragment inhibited the binding to the labeled probe. Previously, we have shown that methylation of the NT/N promoter in vitro resulted in a marked reduction of NT/N promoter activity to baseline levels in transfected liver cancer cell lines (15). Collectively, these results provide additional evidence to support the notion that DNA methylation results in NT/N gene silencing by blocking transcription factor binding to promoter sequences.

Consistent with our findings, other investigators have clearly demonstrated that DNA methylation can repress gene expression in a tissue-specific fashion by inhibiting binding of transcription factors to their cognate sequences (5, 6, 13, 27, 28). For example, Ngo et al. (42) noted that methylation of the rat prolactin and growth hormone promoters correlated with decreased promoter activity. Furthermore, Ianello et al. (27) found that the core promoter of the Pdha-2 gene, which is sufficient for directing testis-specific expression, contains eight CpG dinucleotides; methylation of these sites and, in particular, a CpG site contained within a CREB/ATF site decreased activity of the wild-type promoter by 50%, suggesting that site-specific methylation can modulate gene expression.

Other mechanisms by which methylation can suppress gene expression include inhibition of transcription through methyl-C-binding proteins or by altering chromatin structure, thus influencing gene accessibility (7, 10, 16, 32, 41, 50). To determine whether these latter mechanisms may account for our findings of NT/N expression in colon cancers, we performed locus accessibility assays using the restriction enzymes EcoR I and Msp I. No difference in the digestion patterns of either KM12C or KM20 DNA treated with these enzymes was noted, thus suggesting that these mechanisms of gene suppression do not appear applicable in this particular situation and lending further credence to the role of inhibition of transcription factor binding.

In conclusion, we have shown that DNA methylation plays a role in the silencing of NT/N gene expression in the colon cancer cell line KM20. Treatment with 5-azaC resulted in demethylation of the CpG sites located in the promoter region and resulted in NT/N expression. Methylation of the CpG dinucleotide contained site-blocked protein binding within a distal AP-1 and likely contributes to NT/N gene silencing associated with methylation. On the basis of our findings, we conclude that DNA methylation plays a role in NT/N gene silencing noted in a majority of colorectal cancers. Demethylation may then account for NT/N reexpression identified in ~25% of the cancers. Furthermore, we speculate that DNA methylation, in combination with transcription factors binding the promoter, may play a role in the temporal- and spatial-specific pattern of NT/N gene expression noted in the developing gastrointestinal tract.


    ACKNOWLEDGEMENTS

We thank Eileen Figueroa and Karen Martin for manuscript preparation.


    FOOTNOTES

Z. Dong is a Visiting Scientist at The University of Texas Medical Branch.

This work was supported by grants RO1 AG-10885, RO1 DK-48498, and PO1 DK-35608 from the National Institutes of Health.

Address for reprint requests and other correspondence: B. M. Evers, Dept. of Surgery, The Univ. of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0533 (E-mail: mevers{at}utmb.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.

Received 14 March 2000; accepted in final form 29 June 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Andersson, S, Rosell S, Hjelmquist U, Chang D, and Folkers K. Inhibition of gastric and intestinal motor activity in dogs by (Gln4) neurotensin. Acta Physiol Scand 100: 231-235, 1977[ISI][Medline].

2.   Antequera, F, Macleod D, and Bird AP. Specific protection of methylated CpGs in mammalian nuclei. Cell 58: 509-517, 1989[ISI][Medline].

3.   Baca, I, Feurle GE, Schwab A, Mittmann U, Knauf W, and Lehnert T. Effect of neurotensin on exocrine pancreatic secretion in dogs. Digestion 23: 174-183, 1982[ISI][Medline].

4.   Bean, AJ, Dagerlind A, Hokfelt T, and Dobner PR. Cloning of human neurotensin/neuromedin N genomic sequences and expression in the ventral mesencephalon of schizophrenics and age/sex matched controls. Neuroscience 50: 259-268, 1992[ISI][Medline].

5.   Bednarik, DP, Duckett C, Kim SU, Perez VL, Griffis K, Guenthner PC, and Folks TM. DNA CpG methylation inhibits binding of NF-kappa B proteins to the HIV-1 long terminal repeat cognate DNA motifs. New Biol 3: 969-976, 1991[ISI][Medline].

6.   Ben-Hattar, J, Beard P, and Jiricny J. Cytosine methylation in CTF and Sp1 recognition sites of an HSV tk promoter: effects on transcription in vivo and on factor binding in vitro. Nucleic Acids Res 17: 10179-10190, 1989[Abstract].

7.   Boyes, J, and Bird A. DNA methylation inhibits transcription indirectly via a methyl-CpG binding protein. Cell 64: 1123-1134, 1991[ISI][Medline].

8.   Buschhausen, G, Wittig B, Graessmann M, and Graessmann A. Chromatin structure is required to block transcription of the methylated herpes simplex virus thymidine kinase gene. Proc Natl Acad Sci USA 84: 1177-1181, 1987[Abstract].

9.   Carraway, R, and Leeman SE. The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami. J Biol Chem 248: 6854-6861, 1973[Abstract/Free Full Text].

10.   Cedar, H. DNA methylation and gene activity. Cell 53: 3-4, 1988[ISI][Medline].

11.   Chung, DH, Evers BM, Shimoda I, Townsend CM, Jr, Rajaraman S, and Thompson JC. Effect of neurotensin on gut mucosal growth in rats with jejunal and ileal Thiry-Vella fistulas. Gastroenterology 103: 1254-1259, 1992[ISI][Medline].

12.   Clark, SJ, Harrison J, Paul CL, and Frommer M. High sensitivity mapping of methylated cytosines. Nucleic Acids Res 22: 2990-2997, 1994[Abstract].

13.   Comb, M, and Goodman HM. CpG methylation inhibits proenkephalin gene expression and binding of the transcription factor AP-2. Nucleic Acids Res 18: 3975-3982, 1990[Abstract].

14.   Dignam, JD, Lebovitz RM, and Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11: 1475-1489, 1983[Abstract].

15.   Dong, Z, Wang X, Zhao Q, Townsend CM, Jr, and Evers BM. DNA methylation contributes to expression of the human neurotensin/neuromedin N gene. Am J Physiol Gastrointest Liver Physiol 274: G535-G543, 1998[Abstract/Free Full Text].

16.   Eden, S, and Cedar H. Role of DNA methylation in the regulation of transcription. Curr Opin Genet Dev 4: 255-259, 1994[Medline].

17.   Evers, BM, Ehrenfried JA, Wang X, Townsend CM, Jr, and Thompson JC. Temporal-specific and spatial-specific patterns of neurotensin gene expression in the small bowel. Am J Physiol Gastrointest Liver Physiol 267: G875-G882, 1994[Abstract/Free Full Text].

18.   Evers, BM, Izukura M, Chung DH, Parekh D, Yoshinaga K, Greeley GH, Jr, Uchida T, Townsend CM, Jr, and Thompson JC. Neurotensin stimulates growth of colonic mucosa in young and aged rats. Gastroenterology 103: 86-91, 1992[ISI][Medline].

19.   Evers, BM, Izukura M, Townsend CM, Jr, Uchida T, and Thompson JC. Neurotensin prevents intestinal mucosal hypoplasia in rats fed an elemental diet. Dig Dis Sci 37: 426-431, 1992[ISI][Medline].

20.   Evers, BM, Rajaraman S, Chung DH, Townsend CM, Jr, Wang X, Graves K, and Thompson JC. Differential expression of the neurotensin gene in the developing rat and human gastrointestinal tract. Am J Physiol Gastrointest Liver Physiol 265: G482-G490, 1993[Abstract/Free Full Text].

21.   Evers, BM, Townsend CM, Jr, Upp JR, Allen E, Hurlbut SC, Kim SW, Rajaraman S, Singh P, Reubi JC, and Thompson JC. Establishment and characterization of a human carcinoid in nude mice and effect of various agents on tumor growth. Gastroenterology 101: 303-311, 1991[ISI][Medline].

22.   Evers, BM, Wang X, Zhou Z, Townsend CM, Jr, McNeil GP, and Dobner PR. Characterization of promoter elements required for cell-specific expression of the neurotensin/neuromedin N gene in a human endocrine cell line. Mol Cell Biol 15: 3870-3881, 1995[Abstract].

23.   Evers, BM, Zhou Z, Celano P, and Li J. The neurotensin gene is a downstream target for Ras activation. J Clin Invest 95: 2822-2830, 1995[ISI][Medline].

24.   Evers, BM, Zhou Z, Dohlen V, Rajaraman S, Thompson JC, and Townsend CM, Jr. Fetal and neoplastic expression of the neurotensin gene in the human colon. Ann Surg 223: 464-470, 1996[ISI][Medline].

25.   Feurle, GE, Muller B, and Rix E. Neurotensin induces hyperplasia of the pancreas and growth of the gastric antrum in rats. Gut 28: 19-23, 1987[ISI][Medline].

26.   Frommer, M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, Molloy PL, and Paul CL. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci USA 89: 1827-1831, 1992[Abstract].

27.   Iannello, RC, Young J, Sumarsono S, Tymms MJ, Dahl HH, Gould J, Hedger M, and Kola I. Regulation of Pdha-2 expression is mediated by proximal promoter sequences and CpG methylation. Mol Cell Biol 17: 612-619, 1997[Abstract].

28.   Iguchi-Ariga, SM, and Schaffner W. CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation. Genes Dev 3: 612-619, 1989[Abstract].

29.   Jones, PA. Altering gene expression with 5-azacytidine. Cell 40: 485-486, 1985[ISI][Medline].

30.   Jones, PL, Veenstra GJ, Wade PA, Vermaak D, Kass SU, Landsberger N, Strouboulis J, and Wolffe AP. Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat Genet 19: 187-191, 1998[ISI][Medline].

31.   Kafri, T, Ariel M, Brandeis M, Shemer R, Urven L, McCarrey J, Cedar H, and Razin A. Developmental pattern of gene-specific DNA methylation in the mouse embryo and germ line. Genes Dev 6: 705-714, 1992[Abstract].

32.   Keshet, I, Lieman-Hurwitz J, and Cedar H. DNA methylation affects the formation of active chromatin. Cell 44: 535-543, 1986[ISI][Medline].

33.   Kislauskis, E, and Dobner PR. Mutually dependent response elements in the cis-regulatory region of the neurotensin/neuromedin N gene integrate environmental stimuli in PC12 cells. Neuron 4: 783-795, 1990[ISI][Medline].

34.   Kovesdi, I, Reichel R, and Nevins JR. Role of an adenovirus E2 promoter binding factor in E1A-mediated coordinate gene control. Proc Natl Acad Sci USA 84: 2180-2184, 1987[Abstract].

35.   Lengauer, C, Kinzler KW, and Vogelstein B. DNA methylation and genetic instability in colorectal cancer cells. Proc Natl Acad Sci USA 94: 2545-2550, 1997[Abstract/Free Full Text].

36.   Lewis, JD, Meehan RR, Henzel WJ, Maurer-Fogy I, Jeppesen P, Klein F, and Bird A. Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA. Cell 69: 905-914, 1992[ISI][Medline].

37.   Litt, MD, Hansen RS, Hornstra IK, Gartler SM, and Yang TP. 5-Azadeoxycytidine-induced chromatin remodeling of the inactive X-linked HPRT gene promoter occurs prior to transcription factor binding and gene reactivation. J Biol Chem 272: 14921-14926, 1997[Abstract/Free Full Text].

38.   Morikawa, K, Walker SM, Jessup JM, and Fidler IJ. In vivo selection of highly metastatic cells from surgical specimens of different primary human colon carcinomas implanted into nude mice. Cancer Res 48: 1943-1948, 1988[Abstract].

39.   Morikawa, K, Walker SM, Nakajima M, Pathak S, Jessup JM, and Fidler IJ. Influence of organ environment on the growth, selection, and metastasis of human colon carcinoma cells in nude mice. Cancer Res 48: 6863-6871, 1988[Abstract].

40.   Muraki, K, Mitra SP, Dobner PR, and Carraway RE. Enhanced expression of neurotensin/neuromedin N mRNA and products of NT/NMN precursor processing in neonatal rats. Peptides 14: 1095-1102, 1993[ISI][Medline].

41.   Nan, X, Campoy FJ, and Bird A. MeCP2 is a transcriptional repressor with abundant binding sites in genomic chromatin. Cell 88: 471-481, 1997[ISI][Medline].

42.   Ngo, V, Gourdji D, and Laverriere JN. Site-specific methylation of the rat prolactin and growth hormone promoters correlates with gene expression. Mol Cell Biol 16: 3245-3254, 1996[Abstract].

43.   Okuse, K, Matsuoka I, and Kurihara K. Tissue-specific methylation occurs in the essential promoter element of the tyrosine hydroxylase gene. Brain Res Mol Brain Res 46: 197-207, 1997[ISI][Medline].

44.   Parekh, D, Ishizuka J, Townsend CM, Jr, Haber B, Beauchamp RD, Karp G, Kim SW, Rajaraman S, Greeley G, Jr, and Thompson JC. Characterization of a human pancreatic carcinoid in vitro: morphology, amine and peptide storage, and secretion. Pancreas 9: 83-90, 1994[ISI][Medline].

45.   Reinecke, M. Neurotensin: immunohistochemical localization in central and peripheral nervous system and in endocrine cells and its functional role as neurotransmitter and endocrine hormone. Prog Histochem Cytochem 16: 1-172, 1985[ISI][Medline].

46.   Salvatore, P, Benvenuto G, Caporaso M, Bruni CB, and Chiariotti L. High resolution methylation analysis of the galectin-1 gene promoter region in expressing and nonexpressing tissues. FEBS Lett 421: 152-158, 1998[ISI][Medline].

47.   Schreiber, E, Matthias P, Muller MM, and Schaffner W. Rapid detection of octamer binding proteins with `mini-extracts', prepared from a small number of cells (Abstract). Nucleic Acids Res 17: 6419, 1989[ISI][Medline].

48.   Schwab, M, Alitalo K, Varmus HE, Bishop JM, and George D. A cellular oncogene (c-Ki-ras) is amplified, overexpressed, and located within karyotypic abnormalities in mouse adrenocortical tumour cells. Nature 303: 497-501, 1983[ISI][Medline].

49.   Silverman, AL, Park JG, Hamilton SR, Gazdar AF, Luk GD, and Baylin SB. Abnormal methylation of the calcitonin gene in human colonic neoplasms. Cancer Res 49: 3468-3473, 1989[Abstract].

50.   Tate, PH, and Bird AP. Effects of DNA methylation on DNA-binding proteins and gene expression. Curr Opin Genet Dev 3: 226-231, 1993[Medline].

51.   Thor, K, and Rosell S. Neurotensin increases colonic motility. Gastroenterology 90: 27-31, 1986[ISI][Medline].

52.   Warnecke, PM, and Clark SJ. DNA methylation profile of the mouse skeletal alpha-actin promoter during development and differentiation. Mol Cell Biol 19: 164-172, 1999[Abstract/Free Full Text].


Am J Physiol Gastrointest Liver Physiol 279(6):G1139-G1147
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