DNA methylation contributes to expression of the human neurotensin/neuromedin N gene

Zizheng Dong, Xiaofu Wang, Qingzheng Zhao, Courtney M. Townsend Jr., and B. Mark Evers

Department of Surgery, The University of Texas Medical Branch, Galveston, Texas 77555

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The gut and liver share a common embryological origin. The gene encoding the gut hormone neurotensin/neuromedin N (NT/N) is expressed in the adult small bowel, and NT/N is transiently expressed in the fetal liver, suppressed in the adult liver, and reexpressed in certain liver cancers. In our present study, we found that the NT/N gene was expressed at high levels in the human hepatoma cell line Hep 3B but was not expressed in Hep G2 cells. To further determine the mechanisms regulating NT/N expression, we performed Southern blotting and gene cloning techniques. Neither alteration nor mutation of the NT/N gene was responsible for this differential NT/N expression pattern. Human NT/N promoter constructs were transfected into either Hep 3B or Hep G2. Both cell lines supported NT/N transcription, indicating that the absence of NT/N expression in Hep G2 cells was due to mechanisms other than the absence of positive transcription factors. The role of DNA methylation was next assessed. Methylation of NT/N promoter constructs in vitro resulted in a 67-fold reduction in promoter activity, whereas treatment with the demethylating agent 5-azacytidine induced NT/N expression in Hep G2 cells, thus suggesting that DNA methylation plays a role in the expression of the gut endocrine gene NT/N. Defining the mechanisms regulating NT/N expression in these hepatic-derived cell lines will provide not only a better understanding of cell-specific and developmental regulation of a gut endocrine gene but also possible insight into liver cell lineage patterns and the derivation of certain hepatocellular cancers.

endocrine gene expression; differentiation; hepatocellular cancer

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE HUMAN GASTROINTESTINAL (GI) tract forms during the fourth week of fetal development (44). The endoderm of the primitive gut gives rise to the epithelial lining of the small intestine and colon and the parenchyma of the solid organs (e.g., the liver). This common embryological origin of the liver and gut suggests the presence of shared ancestral stem cells that are capable of multidirectional differentiation. Findings that support this hypothesis include the identification of intestinal markers in hepatoma cell lines (63), the ability of "oval" cells, induced in the rat liver after various chemical and surgical manipulations, to undergo intestinal metaplasia (24, 51, 57, 60), the association of hepatoblastoma with the polyposis coli syndromes (32, 36), and a "hepatoid" differentiation pattern noted in certain colon cancers (28). As fetal development proceeds, however, these stem cells become committed to differentiate into a given cell type with a specialized function. For example, a fixed stem cell population in the proliferating crypts of the small intestine gives rise to four primary cell types (absorptive enterocytes, goblet cells, Paneth cells, and enteroendocrine cells) (11), which express specific genes in a defined pattern along both the vertical and longitudinal gut axes. The molecular basis for this strict tissue-specific pattern of gene expression in the GI tract remains unclear.

Neurotensin (NT), a tridecapeptide originally isolated from bovine hypothalami by Carraway and Leeman (7) and subsequently localized to specialized enteroendocrine cells (N cells) of the adult small bowel (reviewed in Ref. 53), facilitates fatty acid translocation (2), affects gut motility and secretion (53), and stimulates the growth of normal gut mucosa (13, 62) and certain colon and pancreatic cancers (29, 64). In addition, NT enhances hepatocarcinogenesis in rats given N-nitrosomorpholine and augments epidermal growth factor- and transforming growth factor-alpha -mediated DNA synthesis in normal rat hepatocytes (27, 45). We have shown that the gene encoding NT and the structurally related hexapeptide neuromedin N (designated NT/N) are developmentally regulated in the gut of both rats and humans in a distinctive temporal- and spatial-specific pattern (19, 20). NT/N expression is initially low in the fetus but rapidly increases after birth to assume the distinctive adult topographical distribution of increasing NT/N expression along the longitudinal axis of the small bowel. In addition to NT/N expression in the small bowel, we have demonstrated NT/N expression in the fetal human liver; expression was not apparent in the normal adult liver (18). The expression of the gut endocrine gene NT/N in the fetal liver provides yet another example of the close developmental relationship between the liver and the gut.

In our present study, we have cloned the full-length human NT/N cDNA and analyzed the human hepatoma cell lines Hep 3B and Hep G2 to further define mechanisms that contribute to NT/N gene expression. We found that Hep 3B, but not Hep G2, cells express high levels of the NT/N gene; these differences in NT/N expression were not secondary to deletion, mutation, or changes in gene structure. However, Hep G2 cells support NT/N transcription from reporter gene constructs, suggesting that the absence of NT/N expression in this cell line is due to mechanisms other than the absence of positive transcription factors. We next assessed the role of DNA methylation in the control of NT/N expression and found that the activity of enzymatically methylated NT/N reporter constructs was dramatically reduced in Hep G2 cells. Furthermore, treatment of Hep G2 with the demethylating agent 5-azacytidine (5-azaC) resulted in NT/N activation, demonstrating that the suppression of NT/N expression in Hep G2 cells is mediated, at least in part, by DNA methylation. The high-level expression of NT/N in the Hep 3B cell line and the ability of Hep G2 cells to support NT/N transcription provide additional evidence that the liver, at some point during development, expresses what traditionally have been considered "gut-specific" genes. The NT/N gene will provide an important molecular model to further delineate the cellular mechanisms leading to the early differentiation and subsequent maturation of the liver and possible derivation of certain hepatocellular cancers.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell culture and tissue procurement. Hep G2 and Hep 3B, human liver cancer cell lines obtained from American Type Culture Collection (ATCC; Rockville, MD) (1, 35), were cultured in Eagle's minimum essential medium (MEM) with 10% fetal bovine serum (FBS). The human cell line BON, which was established in our laboratory from a pancreatic carcinoid tumor (21, 49), was cultured in Dulbecco's modified Eagle's medium (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. The acquisition and subsequent use of tissues from patients undergoing elective surgical resection at the University of Texas Medical Branch (UTMB) was approved by the Institutional Review Board at UTMB.

RNA extraction, Northern blot, ribonuclease protection, and reverse transcription-polymerase chain reaction. RNA was extracted from tissues by the method of Chomczynski and Sacchi (12) using Ultraspec RNA (Biotecx Laboratories, Houston, TX) according to the manufacturer's suggested protocol (43) and from cell lines as described previously (23) except that digestion with proteinase K was for 1-2 h at 50°C. Poly(A)+ 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, poly(A)+ 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 (3). Hybridization and washing conditions were as described previously (23). Blots were stripped and reprobed with the human albumin gene from ATCC and the constitutively expressed human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene from Ambion (Austin, TX). Ribonuclease (RNase) protection analyses were performed as described previously (23) using the RPA-II kit (Ambion) according to the instructions of the supplier.

For reverse transcription-polymerase chain reaction (RT-PCR), the relative location of the primers used for the RT reaction are shown in Fig. 1. The sequences of the primers are as follows: P1, 5'-GATGATGGCAGGAATGAAAATCCAG-3'; P2, 5'-GTTGAAAAGCCCTGCTGTGACAGA-3'; P3, 5'-GCGAATTCCAAAGGAGGTCGTGCA-3'; P5, 5'-CGCGACTTTGATGTATGCATATTGGTCA-3'; and P8, 5'-TCCCCGCGGACTTGGCTTGTTAGAAGGCT-3'. We incubated 2 µg of total RNA in water at 93°C for 3 min. Samples were quenched on ice, added to 2 µl of 10× PCR buffer, 2 µl dNTP (each at 10 mmol/l), 8 µl MgCl2 (25 mmol/l), 0.5 µl (40 U/µl) RNasin, 0.5 µl oligo(dT)15 (0.5 µg/µl, for P1/P2 amplification) or 0.5 µl P5 (0.5 µg/µl, for P8/P3 amplification) and 15 U avian myeloblastosis virus-reverse transcriptase, and incubated for 1 h at 42°C. In the same tube, 4 µl P1 (50 ng/µl) and P2 (50 ng/µl) (or P8 and P3, each at 50 ng/µl), 1.5 µl GAPDH primer I (4 µM) and GAPDH primer II (4 µM) (both from Clontech), and 2.5 U Taq DNA polymerase were added to the PCR cocktail (final vol 100 µl) and denatured for 8 min at 95°C. The samples were then subjected to 40 cycles of amplification consisting of 1-min denaturation at 94°C, 45-s annealing at 60-65°C, and 1.5-min extension at 72°C, followed by an additional extension step at 72°C for 6 min.


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Fig. 1.   Diagram of polymerase chain reaction (PCR) primers. Diagram showing relative positions in human neurotensin/neuromedin N (NT/N) gene for PCR primers used in analyses shown in Fig. 2C and for cloning human NT/N promoter fragment.

Cloning and sequencing full-length human NT/N cDNA and NT/N promoter region. The sequences of the primers used for cloning the full-length human NT/N cDNA were as follows: dT primer, 5'-AATTGCGGCCCCA(T)17-3'; 5' primer I, 5'-TCCCCGCGGACTTGGCTTGTTAG AAGGCT-3'; and 5' primer II, 5'-TCCCCGGATGATGGCAGGAATGAAAATCCA-3'. We used 1 µg of BON cell poly(A)+ RNA for the RT reaction and PCR as described above. After the first round amplification using the dT primer and 5' primer I, the PCR reaction mixture was diluted to 1 ml and the cDNA fragments were reamplified using 5' primer II, the dT primer, and 3 µl diluted PCR mixture. The PCR product was purified by agarose gel electrophoresis and cloned into the pGEM-T vector (Promega) using 3 U T4 DNA ligase at 8°C for 24 h, and the nucleotide sequence of the cDNA fragment was analyzed as described by Sanger et al. (55) using T7 sequenase (version 2.0).

The sequences of the primers used for cloning the human NT/N promoter region were as follows (primer location is depicted in Fig. 1): P6, 5'-GC<UNL>GGATCC</UNL>GATCTCATGTTTCAC AAAATC-3' (BamH I site is underlined) located at -373 bp; and P3, 5'-GC<UNL>GAATTC</UNL>CAAA GGAGGTCG TGCA-3' (EcoR I site is underlined) located in the first intron. We incubated 0.5-1 µg of genomic DNA in water at 100°C for 15 min, and then it was quenched on ice and added to 10 µl of 10× PCR buffer, 2 µl dNTP (each at 10 mmol/l), 6 µl MgCl2 (25 mmol/l), 4 µl P3 and P6 (each at 50 ng/µl), and 2.5 U Taq DNA polymerase (final vol 100 µl). The samples were then subjected to 30 cycles of amplification consisting of 1-min denaturation at 94°C, 45-s annealing at 63-68°C, and 1.5-min extension at 72°C, followed by an additional extension step at 72°C for 6 min. The PCR products were purified by agarose gel electrophoresis, digested with BamH I and EcoR I restriction enzymes, and cloned into the BamH I/EcoR I site of pUC19. The nucleotide sequences of the cloned fragments were analyzed as described above.

DNA extraction and Southern blot analysis. High molecular weight genomic DNA from the three cell lines was extracted (54) and digested with either Hind III, Bgl II, or Xba I restriction endonucleases. DNA fragments were separated overnight by electrophoresis through a 1% agarose gel with constant voltage (1.5-2.0 V/cm). After electrophoresis, the gel was soaked in 0.25 mol/l HCl for 10-15 min and then rinsed briefly with deionized water. The DNA was denatured, neutralized, and transferred to nitrocellulose filter (54), and then hybridized with the full-length NT/N cDNA probe labeled by a random primer procedure.

Transient transfection, luciferase, and beta -galactosidase assay. The human NT/N reporter plasmid containing the NT/N promoter (-373/+23) cloned upstream of the luciferase gene in Sac I-Sma I-digested pXP1 (48) was from Dr. Paul Dobner (University of Massachusetts, Worcester, MA). Construction of the 5' deletions (-122 and -42) was performed using a PCR-based strategy (Ref. 54 and B. M. Evers, unpublished results). Cells were plated at a density of 0.6 × 106 (Hep G2) or 0.3 × 106 cells/mm2 (Hep 3B) in 60-mm-dishes one day before transfection. NT/N promoter constructs (6-8 µg) were cotransfected with 1.0 µg pCMVbeta (Clontech) by calcium phosphate coprecipitation (for Hep G2) or by lipofectamine from GIBCO-BRL (for Hep 3B). Luciferase and beta -galactosidase assays were performed as described previously (22).

Methylation analyses. The human NT/N promoter construct (-373) and a control plasmid carrying the simian virus 40 promoter and enhancer upstream of the luciferase gene (pSV2Luc) (5) were treated with 13 U of Sss I methylase (New England Biolabs) at 37°C in the presence of 5 mM S-adenosylmethionine for 12 h. Complete methylation of treated plasmids was confirmed by Hpa II restriction enzyme digestion. After 48 h, cellular extracts were assayed for luciferase activity and the transfection efficiency was standardized by beta -galactosidase. Hep G2 or Hep 3B cells were treated with the demethylating agent 5-azaC (Sigma Chemical) at a concentration of 3 or 8 µM and harvested at various time points. RNA was isolated, and RNase protection analyses were performed using the labeled human NT/N probe.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

NT/N gene is constitutively expressed in human hepatoma cell line Hep 3B; the lack of NT/N expression in Hep G2 cells is due most likely to regulation at the level of gene transcription. We have previously shown that NT/N is transiently expressed in the fetal human liver (18). In the present study, we determined whether NT/N is expressed in the hepatic-derived human cell lines Hep 3B and Hep G2 and found, surprisingly, that Hep 3B cells abundantly express the NT/N gene (Fig. 2A). In fact, NT/N expression was as high or higher in Hep 3B cells compared with BON, a cell line that we have previously shown expresses high levels of NT/N mRNA and synthesizes and secretes NT peptide in a fashion identical to the normal intestine (9, 22). In contrast, expression of NT/N was not detected in Hep G2 cells. As controls, the blot was reprobed with human albumin and GAPDH. Both Hep 3B and Hep G2 cells express albumin, as previously demonstrated (1, 35). The constitutively expressed GAPDH gene was noted in all three cell lines.


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Fig. 2.   Analysis of NT/N expression. A: Northern blot analysis of RNA samples [5 µg poly(A)+ RNA] from Hep 3B, Hep G2, and BON cells. Blot was probed with human NT/N probe (pHNT E0.9). Both Hep 3B and BON (positive control) demonstrated NT/N transcripts of the correct sizes (1.5 and 1.0 kb); however, NT/N expression was not apparent in Hep G2 cells. Blot was probed with human albumin gene with expression noted in both Hep 3B and Hep G2 cells but not in BON. Finally, blot was stripped and reprobed with GAPDH to confirm intact RNA and relatively equal loading. B: RNA samples from Hep G2 (lanes 2 and 3), Hep 3B (lanes 4 and 5), and BON cells (lane 6) hybridized with 32P-labeled human NT/N cRNA probe (pHNT E0.9) and analyzed by RNase protection [5 µg poly(A)+ RNA from Hep G2 and Hep 3B used for each lane; 1 µg poly(A)+ BON cell RNA used]. RNA from Hep 3B and BON demonstrate an expected protection product of 180 bases consistent with exon 1 of human NT/N gene; no detectable NT/N mRNA was identified in Hep G2. Lanes 1 and 8, probe alone (without RNase). As a control, tRNA was hybridized with labeled NT/N probe and RNase added; no protection product was noted (lane 7). M, RNA molecular size marker (Ambion). C: total RNA (2 µg) isolated from Hep 3B, BON, and Hep G2 cells was reverse transcribed and PCR-amplified as described in MATERIALS AND METHODS using NT/N primer pairs indicated in Fig. 1. In each experiment, the same reverse transcription (RT) reaction was amplified with GAPDH primers to serve as control for integrity of RT reaction. No template, RT-PCR reaction performed without addition of RNA. M, molecular mass marker.

As confirmation of NT/N expression in Hep 3B cells, RNA was analyzed by RNase protection (Fig. 2B). The expected 180-bp NT/N protection product corresponding to exon 1 was noted in both Hep 3B (Fig. 2B, lanes 4 and 5) and BON cells (Fig. 2B, lane 6) but not in Hep G2 cells (Fig. 2B, lanes 2 and 3). Taken together, these results identify high-level expression of the gut endocrine gene NT/N in the human hepatocellular cancer cell line Hep 3B. In addition, we have recently analyzed a hepatocellular cancer (nonfibrolamellar) resected from a 59-yr-old Asian male. NT/N was expressed in this hepatocellular cancer as noted by RNase protection (data not shown), thus establishing that NT/N is not only expressed in the Hep 3B cell line but also in a hepatocellular cancer in vivo.

In contrast to Hep 3B cells, NT/N was apparently not expressed in Hep G2 cells. We next utilized the more sensitive RT-PCR assay to determine whether Hep G2 cells express NT/N at levels that are not detectable by either Northern blot or RNase protection. In the first analysis, primers P1 and P2, located in the first and third exons respectively, were used to amplify a cDNA fragment of ~380 bp. Using this procedure, we identified the predicted NT/N fragment in both Hep 3B and BON cells; no apparent amplification product was detected in this reaction using RNA from Hep G2 cells (Fig. 2C, left).

In a second assay, primers P8 and P3, located in the 5'-untranslated region of exon 1 and intron 1, respectively, were used to assess whether this absence of NT/N expression occurs at the level of gene transcription. Using this strategy, which was previously described for the thymidine kinase (40) and sucrase isomaltase (42) genes, we identified pre-mRNA transcripts. Although this represents an indirect method of assessing transcription, the absence of unspliced NT/N pre-mRNA in Hep G2 cells would suggest that the NT/N gene is not transcribed. Performance of this RT-PCR reaction yielded a predicted DNA fragment of ~370 bp in both Hep 3B and BON cells but not in Hep G2 cells (Fig. 2C, right). As a control, all reactions contained primers for GAPDH; amplified fragments of ~1.0 kb were detected in all samples (including Hep G2), thus confirming that the RT reaction worked. Because of the increased sensitivity of the RT-PCR, these data provide strong evidence that NT/N is not expressed in Hep G2 cells, and, furthermore, the findings are suggestive that this lack of expression is a result of transcriptional regulation.

The NT/N gene is intact in both Hep 3B and Hep G2 cells. Possible explanations for the absence of NT/N expression in the Hep G2 cell line include chromosomal deletions or mutations resulting in a defect of the NT/N gene itself. Bean et al. (3) previously described a human NT/N genomic clone encompassing exons 1 through 3; however, exon 4, which encodes the NT and neuromedin N peptides, was not included in this partial NT/N clone. To accurately assess the entire coding region of the human NT/N gene, we cloned the full-length human NT/N cDNA to utilize as a probe for Southern blots.

Cloning the full-length cDNA was accomplished using BON cell RNA as a template, oligo(dT)17 as a 3'-end primer, and a 5'-end gene-specific primer in a two-round amplification similar to a nested PCR reaction (25). Nucleotide sequence analysis of the positive clones demonstrated a cDNA fragment of 756 bp that, similar to the canine NT/N prepropeptide (16), contains an open reading frame encoding a predicted precursor protein of 170 amino acid residues (Fig. 3A). In contrast, the precursor NT/N peptide in cows (8) and rats (33) is composed of 169 amino acids. Of the 169 comparable positions, the human NT/N protein is 90% identical to that of both dogs and cows and 78% identical to rat NT/N sequences (Fig. 3B). At the nucleotide level, the human NT/N precursor is 92%, 91%, and 81% identical to the dog, cow, and rat nucleotide sequences, respectively. Taken together, these results indicate a high degree of conservation of the NT/N coding region among the four species.


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Fig. 3.   Cloning full-length human NT/N cDNA and analysis of genomic DNA. A: composite sequence determined from human full-length cDNA clone and predicted amino acid sequence of human preproneurotensin/neuromedin N. The neuromedin N and NT coding regions, located in tandem on exon 4, are boxed by solid lines. B: comparison of the putative amino acid sequence of human NT/N to known sequences of cow, dog, and rat; -, identical sequences; +, similar sequences. C: genomic DNA (5 µg) from Hep 3B, BON, and Hep G2 cells was digested with Xba I (X), Hind III (H), or Bgl II (B) and transferred to nitrocellulose and hybridized with full-length NT/N cDNA probe.

This full-length clone was then labeled and used to probe genomic DNA extracted from Hep G2, Hep 3B, and BON cells. The pattern of hybridization was the same in all three cell lines (Fig. 3C), suggesting that the NT/N gene was present and that there was no gross rearrangement in the coding region. Next, PCR was used to analyze the proximal NT/N promoter region. DNA extracted from the cell lines was amplified, and the resulting fragment was subcloned and sequenced. The sequence of the proximal NT/N promoter was identical in all three cell lines and the same as previously published for the human NT/N gene (3) (data not shown).

Both Hep 3B and Hep G2 cells possess the requisite cellular factors to transcribe the NT/N gene. Previously, we have shown that the proximal promoter region (216 bp upstream from the transcriptional start site) of the rat NT/N gene is sufficient to direct high-level expression in the BON endocrine cell line (22). This proximal promoter region, which is highly conserved in the human NT/N gene (3), contains a crucial AP-1/CRE site located ~40 bp from the transcriptional start site. Other more distal regions include near-consensus CRE and AP-1 sites that are important for NT/N induction in PC12 cells (34) but are not as important for constitutive NT/N expression in BON.

We next determined whether Hep 3B or Hep G2 cells could support transcription of human NT/N promoter constructs. A -373 promoter construct contains the proximal AP-1/CRE site and more distal CRE and AP-1 sites (3); the -122 deletion construct contains the proximal AP-1/CRE site, and the -42 construct contains the NT/N TATA box. Transient transfections into Hep 3B cells resulted in high-level NT/N activity for both the -373 and -122 promoter deletions compared with the promoterless control vector pXP1; the NT/N promoter was silenced with deletion to -42 (Fig. 4A). Surprisingly, transfection of the -373 NT/N construct into Hep G2 cells resulted in NT/N promoter activity similar to that noted in the NT/N-expressing Hep 3B cell line (Fig. 4B). Also similar to Hep 3B, deletion to -42 resulted in near silencing of the NT/N promoter in Hep G2 cells. Deletion to -122 resulted in a greater decrease in NT/N promoter activity in Hep G2 compared with Hep 3B cells. The significance of this finding is not known but may indicate the importance of the near consensus CRE and AP-1 sites for NT/N promoter activity in Hep G2 cells. Taken together, our results demonstrate a disparity between expression of NT/N mRNA in the two liver-derived cell lines and the ability of the cells to transcribe from the NT/N promoter. These findings suggest that the requisite cellular machinery to transcribe the NT/N gene is present in both cell lines; however, other mechanisms are responsible for NT/N gene suppression in Hep G2 cells.


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Fig. 4.   Transfection of chimeric NT/N gene constructs into Hep 3B (A) and Hep G2 (B) cells. Luciferase activity (expressed as fold activation compared with pXP1 promoterless plasmid) is means ± SD of 5 independent transfections (Hep 3B) and 7 independent transfections (Hep G2) after normalization for beta -galactosidase expression using 1 µg pCMVbeta (Clontech).

NT/N gene suppression in Hep G2 cells is mediated, in part, by gene methylation. The possible role that gene methylation plays in the expression of NT/N in these two cell lines was next assessed. As a first step, methylation of the NT/N promoter (-373/+26) construct in vitro with the CpG methylase Sss I was performed, and the methylated constructs were transfected into Hep G2 cells. Results were compared with methylation of a pSV2Luc control vector. The completeness of CpG methylation was verified by digesting with Hpa II with fully methylated plasmids resistant to Hpa II digestion (Fig. 5A). CpG methylation of the human NT/N promoter produced an ~67-fold reduction of transcriptional activity (Fig. 5B). In contrast, similar to the findings of others (4, 26), the simian virus 40 enhancer is only weakly sensitive to methylation as noted by only a threefold reduction in transcriptional activity of the pSV2Luc control vector (Fig. 5B). Therefore, these results demonstrate that transcriptional activity of the human NT/N gene is downregulated by DNA methylation in a preferential and dramatic fashion. This repression was not simply due to methylation of the luciferase gene or of plasmid DNA, since the relatively methylation-insensitive SV40 enhancer and promoter was much less affected by the same treatment.


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Fig. 5.   In vitro methylation. A: -373 NT/N and pSV2Luc were methylated with Sss I methylase. Both Sss I-treated (+) and untreated (-) plasmid DNAs were linearized with Hind III (H) and challenged with methylation-sensitive restriction endonuclease Hpa II (Hp) to test for completeness of methylation. B: methylated or unmethylated plasmids (5 µg) were transfected into Hep G2 cells. Luciferase activities for methylated plasmids are shown as %unmethylated constructs. Relative luciferase activities were as follows: 1,991 ± 593 and 29.8 ± 26.3 for unmethylated vs. methylated -373 NT/N, respectively; 3,898 ± 142 and 1,150 ± 474.7 for unmethylated vs. methylated pSV2Luc, respectively. Values represent means ± SD from 3 independent experiments.

The role of methylation in regulating NT/N gene expression was further assessed by treating Hep G2 cells with the demethylating agent 5-azaC (Fig. 6A). Cells treated with 3 µM 5-azaC for 4 days demonstrated a low level of NT/N expression (Fig. 6A, lanes 3 and 4) compared with untreated Hep G2 cells (Fig. 6A, lane 2). Addition of 8 µM 5-azaC for 4 days (Fig. 6A, lanes 5 and 6) resulted in higher NT/N expression compared with 3 µM 5-azaC (Fig. 6A, lanes 3 and 4). Finally, cells were treated with 8 µM 5-azaC for 4 days, at which time the medium was changed to fresh medium with 3 µM 5-azaC for an additional 4 days (Fig. 6A, lane 7). NT/N was expressed, but at lower levels than with treatment with 8 µM alone for 4 days. BON cell RNA (Fig. 6A, lane 8) was added as a positive control and demonstrated high-level NT/N expression. A separate RNase protection gel was performed, using the human GAPDH probe to confirm that RNA samples were intact (Fig. 6B). Treatment of Hep 3B cells with 5-azaC produced no apparent changes in NT/N expression levels (data not shown). Taken together, the reduction of NT/N promoter activity by methylation and the induction of NT/N expression by 5-azaC demonstrate that DNA methylation plays a role in NT/N gene suppression in Hep G2 cells.


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Fig. 6.   Effect of 5-azacytidine (5-azaC) on NT/N expression in Hep G2 cells. A: RNA [10 µg poly(A)+ RNA] from untreated Hep G2 cells (lane 2) or Hep G2 cells treated with various concentrations of demethylating agent 5-azaC and analyzed by RNase protection. Lane 3, 3 µM 5-azaC for 4 days; lane 4, 3 µM 5-azaC for 2 days then changed to fresh medium with 3 µM 5-azaC for additional 2 days; lane 5, 8 µM 5-azaC for 4 days; lane 6, 8 µM 5-azaC for 2 days then changed to fresh medium with 8 µM 5-azaC for additional 2 days; lane 7, 8 µM 5-azaC for 4 days then changed to medium containing 3 µM 5-azaC for an additional 4 days. Lane 8, BON cell RNA [0.5 µg poly(A)+ RNA]. Lane 1, probe alone. M, RNA molecular size marker. B: to confirm intact RNA, a separate RNase protection analysis was performed using a human GAPDH probe (Ambion). Lanes 2-7 correspond to the Hep G2 treatment groups shown in A. Lane 8, BON cell RNA hybridized with GAPDH. Lane 1, GAPDH probe alone.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the present study, we demonstrate high-level expression of the terminally differentiated endocrine gene NT/N in the human hepatoma cell line Hep 3B. In fact, expression levels of NT/N by Northern blot were as high in Hep 3B cells as in the BON endocrine cell line. Earlier reports have described NT/N gene expression in the rare fibrolamellar hepatic tumor (14, 18, 52), which is characterized by a different morphology, no causal association with the hepatitis virus, and an overall better prognosis than the more common hepatocellular cancer (reviewed in Ref. 15). Our results, however, suggest that NT/N gene expression is not unique to the fibrolamellar variant and can also be associated with other hepatocellular cancers.

Expression of the NT/N gene may provide insight into the particular cell lineage that gives rise to the hepatocellular cancer. A prevailing hypothesis is that tumor cells are transformed stem cells or early precursor cells (50, 51, 56, 57). Therefore, the expression of NT/N may not simply reflect activation or induction by a transformation event but rather a normal gene that is representative of an immature cell population. Consistent with this notion is the finding of NT/N gene expression in the human liver during early fetal development (18). Although Wang et al. (61) have shown that gastrin, another gut hormone, is expressed in the liver of transgenic mice containing the human gastrin minigene, our results are, to our knowledge, the only demonstration of expression of a gut endocrine gene in not only the normal fetal liver but also hepatocellular cancers. Thus it is clear that the liver can support transcription of gut genes traditionally regarded as localized only to enteroendocrine cells.

In marked contrast to in Hep 3B cells, increasingly sensitive techniques failed to detect NT/N expression in untreated Hep G2 cells. Both Hep 3B and Hep G2 hepatoma cells are epithelial in morphology and retain the capacity to synthesize plasma proteins including albumin, but differ in that Hep 3B cells synthesize and secrete the hepatitis B surface antigen and produce tumors when injected into athymic nude mice (1, 35). At least three general possibilities exist to explain silencing of gene expression: 1) gene mutation or deletion, 2) a lack of necessary cellular factors to activate transcription (i.e., positive-acting transcription proteins), and 3) mechanisms that block transcription (e.g., gene methylation or repressor proteins that bind the promoter) (17, 47, 59). The absence of NT/N gene expression in Hep G2 cells was not due to an apparent deletion of the gene, alteration in the gene structure, or mutation of the promoter region as demonstrated by Southern blotting and gene cloning techniques. Moreover, Hep G2 cells support NT/N transcription from reporter gene constructs, suggesting that these cells possess the necessary factors to activate the NT/N promoter. Therefore, our results suggest that the absence of NT/N expression in Hep G2 cells may be due to mechanisms that can block gene transcription.

In the present study, we demonstrate by independent and complementary approaches that DNA methylation plays a role in NT/N gene suppression in Hep G2 cells. Partial activation of the suppressed NT/N gene was induced by short-term treatment of Hep G2 cells with the demethylating agent 5-azaC. Moreover, in vitro methylation of the NT/N promoter sequences markedly inhibited NT/N activity in Hep G2 cells. Although it is becoming increasingly evident that DNA methylation ensures the silencing of certain tissue-specific genes in nonexpressing cells (4, 30, 37, 41, 47, 58, 59), this is the first demonstration that methylation may play a role in the expression of a gut endocrine gene. Previously, we have shown that the high-level NT/N expression noted in BON cells is dependent on a crucial AP-1/CRE proximal promoter element that binds both AP-1 and CREB/ATF proteins (22). Therefore, one possibility is that DNA methylation interferes with NT/N expression by affecting the binding of these transcription factors to the NT/N proximal promoter. Another possibility is that DNA methylation can inhibit transcription through methyl-C-binding proteins that bind specifically to methylated, but not unmethylated, DNA (46, 59). Finally, DNA methylation could alter the chromatin structure, thus influencing gene accessibility (31, 59). Although we did not specifically address the question of which of these mechanisms is involved in NT/N gene regulation, the results of our present study using the transiently transfected methylated plasmids would argue in favor of one of the first two mechanisms.

Gene methylation appears to be involved in several crucial cellular processes, including differentiation, development, and carcinogenesis (10, 30, 38, 39, 59). The differences in NT/N gene expression in Hep 3B and Hep G2 cells suggest derivation of these lines from different cell lineages in the liver or, alternatively, may represent different levels of cellular maturation or differentiation. The results from our present study suggest that DNA methylation plays a role in NT/N gene regulation; however, gene methylation alone does not fully account for the marked suppression of NT/N in Hep G2 cells. We suspect that the strict tissue-specific regulation of NT/N gene expression is dependent on the combinatorial effects of not only gene methylation but also enhancer and/or repressor proteins that bind to the proximal NT/N promoter (22) and possible additional sites outside of the immediate 5'-flanking region (e.g., the first intron). Further studies should provide important insight regarding the interplay between DNA methylation and the complex network of ubiquitous and tissue-limited transcription factors that govern differentiation and the specific developmental expression pattern of NT/N in the gut and liver. In addition, these hepatic-derived cell lines, which differ greatly in their ability to express NT/N, should provide useful models to better characterize the mechanisms regulating cell-specific expression of the NT/N gene.

In conclusion, the important points from our present study include 1) the identification of NT/N gene expression in hepatocellular cancers, which, in combination with our previous findings of expression in the fetal liver, further emphasizes that the liver can support transcription of genes traditionally regarded as only localized to certain cells of the gut; 2) the cloning of the entire human NT/N cDNA, including exon 4, which encodes both the NT and neuromedin N peptides; and 3) the demonstration that DNA methylation contributes to the regulation of expression of a gut endocrine gene. Finally, the NT/N gene will not only provide a useful model to delineate mechanisms contributing to gut development but may also be useful in determining liver cell lineage patterns and derivation of certain hepatocellular cancers.

    ACKNOWLEDGEMENTS

We thank Drs. E. Aubrey Thompson and Mark R. Hellmich for advice and critical reading of the manuscript and Dr. Paul Dobner for continued advice and assistance during the course of our studies. We also thank Dr. Tien C. Ko for providing samples of a hepatocellular cancer after liver resection. The NT/N deletion plasmids -122 and -42 were constructed by Dr. Thomas G. Wood in the Recombinant DNA Core Facility of The Sealy Center for Molecular Science. In addition, we thank Karen Martin and Eileen Figueroa for manuscript preparation.

    FOOTNOTES

The sequences reported here have been deposited in the GenBank data base (accession no. U91618).

This work was supported by National Institutes of Health Grants DK-48498, AG-10885, and DK-35608 and the James E. Thompson Memorial Foundation.

Z. Dong was on leave from the Institute of Biotechnology, Beijing, 100071, People's Republic of China.

Present address of Q. Zhao: Dept. of Biochemistry, Cancer Institute, Chinese Academy of Medical Sciences, Beijing 100021, People's Republic of China.

Address for reprint requests: B. M. Evers, Dept. of Surgery, The Univ. of Texas Medical Branch, 301 University Blvd., Galveston, Texas 77555.

Received 7 October 1997; accepted in final form 8 December 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Aden, D. P., A. Fogel, S. Plotkin, I. Damjanov, and B. B. Knowles. Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line. Nature 282: 615-616, 1979[Medline].

2.   Armstrong, M. J., M. C. Parker, C. F. Ferris, and S. E. Leeman. Neurotensin stimulates [3H]oleic acid translocation across rat small intestine. Am. J. Physiol. 251 (Gastrointest. Liver Physiol. 14): G823-G829, 1986[Medline].

3.   Bean, A. J., J. A. Dagerlind, T. Hökfelt, and P. R. Dobner. 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[Medline].

4.   Benvenuto, G., M. L. Carpentieri, P. Salvatore, L. Cindolo, C. B. Bruni, and L. Chiariotti. Cell-specific transcriptional regulation and reactivation of galectin-1 gene expression are controlled by DNA methylation of the promoter region. Mol. Cell. Biol. 16: 2736-2743, 1996[Abstract].

5.   Brasier, A. R., J. E. Tate, and J. F. Habener. Optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines. Biotechniques 7: 1116-1122, 1989[Medline].

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

8.   Carraway, R., and S. E. Leeman. The amino acid sequence of a hypothalamic peptide, neurotensin. J. Biol. Chem. 250: 1907-1911, 1975[Abstract].

9.   Carraway, R. E., S. P. Mitra, B. M. Evers, and C. M. Townsend, Jr. BON cells display the intestinal pattern of neurotensin/neuromedin N precursor processing. Regul. Pept. 53: 17-29, 1994[Medline].

10.   Cedar, H., and A. Razin. DNA methylation and development. Biochim. Biophys. Acta 1049: 1-8, 1990[Medline].

11.   Cheng, H., and C. P. Leblond. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian theory of the origin of the four epithelial cell types. Am. J. Anat. 141: 537-561, 1974[Medline].

12.   Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

13.   Chung, D. H., B. M. Evers, I. Shimoda, C. M. Townsend, Jr., S. Rajaraman, and J. C. Thompson. Effect of neurotensin on gut mucosal growth in rats with jejunal and ileal Thiry-Vella fistulas. Gastroenterology 103: 1254-1259, 1992[Medline].

14.   Collier, N. A., K. Weinbren, S. R. Bloom, Y. C. Lee, H. J. F. Hodgson, and L. H. Blumgart. Neurotensin secretion by fibrolamellar carcinoma of the liver. Lancet 1: 538-540, 1984[Medline].

15.   Craig, J. R. Fibrolamellar carcinoma: clinical and pathological features. In: Liver Cancer, edited by K. Okuda, and E. Tabor. London: Churchill-Livingston, 1997.

16.   Dobner, P. R., D. L. Barber, L. Villa-Komaroff, and C. McKiernan. Cloning and sequence analysis of cDNA for the canine neurotensin/neuromedin N precursor. Proc. Natl. Acad. Sci. USA 84: 3516-3520, 1987[Abstract].

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

18.   Ehrenfried, J. A., Z. Zhou, J. C. Thompson, and B. M. Evers. Expression of the neurotensin gene in fetal human liver and fibrolamellar carcinoma. Ann. Surg. 220: 484-491, 1994[Medline].

19.   Evers, B. M., J. A. Ehrenfried, X. Wang, C. M. Townsend, Jr., and J. C. Thompson. Temporal-specific and spatial-specific patterns of neurotensin gene expression in the small bowel. Am. J. Physiol. 267 (Gastrointest. Liver Physiol. 30): G875-G882, 1994[Abstract/Free Full Text].

20.   Evers, B. M., S. Rajaraman, D. H. Chung, C. M. Townsend, Jr., X. Wang, K. Graves, and J. C. Thompson. Differential expression of the neurotensin gene in the developing rat and human gastrointestinal tract. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28): G482-G490, 1993[Abstract/Free Full Text].

21.   Evers, B. M., C. M. Townsend, Jr., J. R. Upp, Jr., E. Allen, S. C. Hurlbut, S. W. Kim, S. Rajaraman, P. Singh, J. C. Reubi, and J. C. Thompson. Establishment and characterization of a human carcinoid in nude mice and effect of various agents on tumor growth. Gastroenterology 101: 303-311, 1991[Medline].

22.   Evers, B. M., X. Wang, Z. Zhou, C. M. Townsend, Jr., G. P. McNeil, and P. R. Dobner. 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, B. M., Z. Zhou, P. Celano, and J. Li. The neurotensin gene is a downstream target for Ras activation. J. Clin. Invest. 95: 2822-2830, 1995[Medline].

24.   Fausto, N. Hepatocyte differentiation and liver progenitor cells. Curr. Opin. Cell Biol. 2: 1036-1042, 1990[Medline].

25.   Frohman, M. A., M. K. Dush, and G. R. Martin. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85: 8998-9002, 1988[Abstract].

26.   Graessmann, M., A. Graessmann, H. Wagner, H. Werner, and D. Simon. Complete DNA methylation does not prevent polyoma and simian virus 40 virus early gene expression. Proc. Natl. Acad. Sci. USA 80: 6470-6474, 1983[Abstract].

27.   Hasegawa, K., and B. I. Carr. Neurotensin-amplification of DNA synthesis stimulated by EGF or TGF alpha  in primary cultures of adult rat hepatocytes. Cell Struct. Funct. 18: 105-110, 1993[Medline].

28.   Hocking, G. R., M. Shembrey, D. Hay, and A. G. Östör. alpha -Fetoprotein-producing adenocarcinoma of the sigmoid colon with possible hepatoid differentiation. Pathology 27: 277-279, 1995[Medline].

29.   Iwase, K., B. M. Evers, M. R. Hellmich, H. J. Kim, S. Higashide, D. Gully, J. C. Thompson, and C. M. Townsend, Jr. Inhibition of neurotensin-induced pancreatic cancer growth by a nonpeptide neurotensin receptor antagonist, SR48692. Cancer 79: 1787-1793, 1997[Medline].

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

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

32.   Kingston, J. E., A. Herbert, G. J. Draper, and J. R. Mann. Association between hepatoblastoma and polyposis coli. Arch. Dis. Child. 58: 959-962, 1983[Abstract].

33.   Kislauskis, E., B. Bullock, S. McNeil, and P. R. Dobner. The rat gene encoding neurotensin and neuromedin N structure, tissue-specific expression, and evolution of exon sequences. J. Biol. Chem. 263: 4963-4968, 1988[Abstract/Free Full Text].

34.   Kislauskis, E., and P. R. Dobner. 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[Medline].

35.   Knowles, B. B., C. C. Howe, and D. P. Aden. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science 209: 497-499, 1980[Medline].

36.   Krush, A. J., E. I. Traboulsi, J. A. Offerhaus, I. H. Maumenee, J. H. Yardley, and L. S. Levin. Hepatoblastoma, pigmented ocular fundus lesions and jaw lesions in Gardner syndrome. Am. J. Med. Genet. 29: 323-332, 1988[Medline].

37.   Kudo, S., and M. Fukuda. Tissue-specific transcriptional regulation of human leukosialin (CD43) gene is achieved by DNA methylation. J. Biol. Chem. 270: 13298-13302, 1995[Abstract/Free Full Text].

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

39.   Li, E., T. H. Bestor, and R. Jaenisch. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69: 915-926, 1992[Medline].

40.   Lipson, K. E., and R. Baserga. Transcriptional activity of the human thymidine kinase gene determined by a method using the polymerase chain reaction and an intron-specific probe. Proc. Natl. Acad. Sci. USA 86: 9774-9777, 1989[Abstract].

41.   Lu, S., and P. J. A. Davies. Regulation of the expression of the tissue transglutaminase gene by DNA methylation. Proc. Natl. Acad. Sci. USA 94: 4692-4697, 1997[Abstract/Free Full Text].

42.   Markowitz, A. J., G. D. Wu, A. Bader, Z. Cui, L. Chen, and P. G. Traber. Regulation of lineage-specific transcription of the sucrase-isomaltase gene in transgenic mice and cell lines. Am. J. Physiol. 269 (Gastrointest. Liver Physiol. 32): G925-G939, 1995[Abstract/Free Full Text].

43.   Mehra, M. RNA isolation from cells and tissues. In: A Laboratory Guide to RNA: Isolation, Analysis, and Synthesis, edited by P. A. Krieg. New York: Wiley-Liss, 1996, p. 1-20.

44.  Moore, K. L., and T. V. N. Persaud (Editors). The digestive system. In: The Developing Human: Clinically Oriented Embryology. Philadelphia, PA: Saunders, 1993, p. 237-264.

45.   Nakaizumi, A., H. Uehara, M. Baba, H. Iishi, and M. Tatsuta. Enhancement by neurotensin of hepatocarcinogenesis by N-nitrosomorpholine in Sprague-Dawley rats. Cancer Lett. 110: 57-61, 1996[Medline].

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

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

48.   Nordeen, S. K. Luciferase reporter gene vectors for analysis of promoters and enhancers. Biotechniques 6: 454-458, 1988[Medline].

49.   Parekh, D., J. Ishizuka, C. M. Townsend, Jr., B. Haber, R. D. Beauchamp, G. Karp, S. W. Kim, S. Rajaraman, G. H. Greeley, Jr., and J. C. Thompson. Characterization of a human pancreatic carcinoid in vitro: morphology, amine and peptide storage and secretion. Pancreas 9: 83-90, 1994[Medline].

50.   Pierce, G. B. Neoplasms, differentiations, and mutations. Am. J. Pathol. 77: 103-118, 1974[Medline].

51.   Ponder, K. P. Analysis of liver development, regeneration, and carcinogenesis by genetic marking studies. FASEB J. 10: 673-684, 1996[Abstract/Free Full Text].

52.   Read, D., A. Shulkes, R. Fernley, and R. Simpson. Characterization of neurotensin(6-13) from an hepatic fibrolamellar carcinoma. Peptides 12: 887-892, 1991[Medline].

53.   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[Medline].

54.   Sambrook, J., E. F. Fritsch, and T. Maniatis (Editors). Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor, New York: Cold Spring Harbor Press, 1989.

55.   Sanger, F., S. Niklen, and A. R. Couloson. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467, 1977[Abstract].

56.   Sell, S., and H. A. Dunsford. Evidence for the stem cell origin of hepatocellular carcinoma and cholangiocarcinoma. Am. J. Pathol. 134: 1347-1363, 1989[Abstract].

57.   Sigal, S. H., S. Brill, A. S. Fiorino, and L. M. Reid. The liver as a stem cell and lineage system. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G139-G148, 1992[Abstract/Free Full Text].

58.   Späth, G. F., and M. C. Weiss. Hepatocyte nuclear factor 4 expression overcomes repression of the hepatic phenotype in dedifferentiated hepatoma cells. Mol. Cell. Biol. 17: 1913-1922, 1997[Abstract].

59.   Tate, P. H., and A. P. Bird. Effects of DNA methylation on DNA-binding proteins and gene expression. Curr. Opin. Genet. Dev. 3: 226-231, 1993[Medline].

60.   Tatematsu, M., T. Kaku, A. Medline, and E. Farber. Intestinal metaplasia as a common option of oval cells in relation to cholangiofibrosis in liver of rats exposed to 2-acetylaminofluorene. Lab. Invest. 52: 354-362, 1985[Medline].

61.   Wang, T. C., T. J. Koh, A. Varro, R. J. Cahill, C. A. Dangler, J. G. Fox, and G. J. Dockray. Processing and proliferative effects of human progastrin in transgenic mice. J. Clin. Invest. 98: 1918-1929, 1996[Abstract/Free Full Text].

62.   Wood, J. G., H. D. Hoang, L. J. Bussjaeger, and T. E. Solomon. Neurotensin stimulates growth of small intestine in rats. Am. J. Physiol. 255 (Gastrointest. Liver Physiol. 18): G813-G817, 1988[Abstract/Free Full Text].

63.   Yoshida, Y., A. Kaneko, N. Chisaka, and T. Onoe. Appearance of intestinal type of tumor cells in hepatoma tissue induced by 3-methyl-4-dimethylaminoazobenzene. Cancer Res. 38: 2753-2758, 1978[Abstract].

64.   Yoshinaga, K., B. M. Evers, M. Izukura, D. Parekh, T. Uchida, C. M. Townsend, Jr., and J. C. Thompson. Neurotensin stimulates growth of colon cancer. Surg. Oncol. 1: 127-134, 1992[Medline].


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