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
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ABSTRACT |
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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
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INTRODUCTION |
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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-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.
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MATERIALS AND METHODS |
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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 [
-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.
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RESULTS |
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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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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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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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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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We thank Eileen Figueroa and Karen Martin for manuscript preparation.
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FOOTNOTES |
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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.
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