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[Cancer Research 61, 7722-7726, November 1, 2001]
© 2001 American Association for Cancer Research

Advances in Brief

Up-Regulation of the Ectodermal-Neural Cortex 1 (ENC1) Gene, a Downstream Target of the ß-Catenin/T-Cell Factor Complex, in Colorectal Carcinomas1

Manabu Fujita, Yoichi Furukawa, Tatsuhiko Tsunoda, Toshihiro Tanaka, Michio Ogawa and Yusuke Nakamura2

Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639 [M. F., Y. F., Y. N.]; Department of Surgery II, Kumamoto University School of Medicine, Kumamoto 860-8556 [M. F., M. O.]; SNP Research Center, Riken (Institute of Physical and Chemical Research), Tokyo 108-8639 [T. Ts., T. Ta.], Japan


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
To clarify the molecular mechanisms of human carcinogenesis associated with abnormal Wnt/wingless signaling, we searched for genes the expression of which was significantly altered by introduction of wild-type AXIN1 into LoVo colon cancer cells. By means of a cDNA microarray, we compared expression profiles of LoVo cells infected with either adenoviruses expressing wild-type AXIN1 (Ad-Axin) or those expressing a control gene (Ad-LacZ). Among the genes showing altered expression, the ectodermal-neural cortex 1 (ENC1) gene was down-regulated in response to Ad-Axin. The promoter activity of ENC1 was elevated ~3-fold by transfection of an activated form of ß-catenin together with wild-type T-cell factor (Tcf)4 in HeLa cells. Semiquantitative reverse transcription-PCR experiments revealed that expression of ENC1 was increased in more than two-thirds of 24 primary colon cancer tissues that we examined compared with corresponding noncancerous mucosae. Introduction of exogenous ENC1 increased the growth rate of HCT116 colon cancer cells in serum-depleted medium. In other experiments, overexpression of ENC1 in HT-29 colon cancer cells suppressed the usual increase of two differentiation markers, in response to treatment with sodium butyrate, a differentiation-inducible agent. These data suggest that ENC1 is regulated by the ß-catenin/Tcf pathway and that its altered expression may contribute to colorectal carcinogenesis by suppressing differentiation of colonic cells.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Genes in the Wnt/wingless signaling pathway play critical roles in differentiation and morphogenesis during embryogenesis (1) . Recent progress in cancer research has underscored the importance of this signal-transduction pathway in human tumors, whether arising in the colon, liver, prostate, stomach, brain, endometrium, or elsewhere (2) . ß-catenin, a major element in Wnt/wingless signaling, also plays a pivotal role in cell-to-cell adhesion together with E-cadherin (3) . Abnormal intracellular accumulation of ß-catenin as a consequence of genetic alterations in the adenomatous polyposis coli (APC), Axin (AXIN1), or ß-catenin (CTNNB1) genes has been observed in various human cancers including colorectal and hepatocellular carcinomas (4) . Accumulated ß-catenin interacts with Tcf3 /LEF and translocates to the nucleus, in which it transactivates target genes including c-myc, cyclin D1, matrilysin, c-jun, fra-1, NBL4, and MARKL1 (5, 6, 7, 8, 9, 10, 11) . Axin, an inhibitor of this pathway, functions as a scaffold protein of ß-catenin and glycogen synthase kinase-3ß (GSK-3ß), and facilitates the degradation of ß-catenin via the ubiquitin-proteosome system (4) . Adenovirus-mediated gene transfer of wild-type AXIN1 can induce apoptosis not only in Axin-deficient cells but also in cells with mutated ß-catenin or APC (12) .

ENC1 was isolated as a gene encoding an actin-associated protein that is expressed in the neuroectodermal region of the epiblast during early gastrulation, and later in the nervous system (13) . Kim et al. (14) identified cDNAs that encode ENC1, which they termed nuclear-restricted protein/brain (NRP/B), and found that NRP/B was involved in neuronal differentiation, being expressed abundantly in the brain and localized in nuclei. The deduced ENC1 protein contains two major structural elements, a broad complex Tramtrack bric-a-brac/Pox virus and zinc finger (BTB/POZ) domain-like structure in the NH2 terminus and six copies of "kelch motif" repeats in the COOH-terminal region (14) . In vitro, the BTB/POZ domain mediates protein-protein interactions by forming both dimers and heterodimers (15) . Members of the kelch family are important for cytoskeletal organization and function in several species (16) . When expressed in the brain, ENC1 appears to interact with the actin cytoskeleton in the cytoplasm through repeats of the kelch motif (17) . Expression of ENC1 was reportedly up-regulated during neuronal differentiation in murine Neuro2A cells and human SH-SY5Y neuroblastoma cells (14) , and it was induced during adipocyte differentiation in preadipocyte cell line 3T3-L1 (17) . Moreover, expression of ENC1 is frequently elevated in brain tumors (18) .

In the study reported here, we demonstrated that expression of ENC1 was down-regulated in response to transduction of wild-type AXIN1 into colon cancer cells, and that its expression was often elevated in primary colon cancers. We discuss further the potential role of ENC1 in colorectal tumorigenesis.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Cell Lines and Clinical Materials.
Human colon cancer cell lines SW480, LoVo, HT29, and HCT116 and a human cervix cancer cell line HeLa were obtained from the American Type Culture Collection (ATCC, Rockville, MD). All of the cells were cultured as monolayers in appropriate media, as follows: Leibovitz’s L-15 (Life Technologies, Inc., Grand Island, NY) for SW480; HAM F-12 nutrient mixture (Life Technologies, Inc.) for LoVo; McCoy’s medium (Life Technologies, Inc.) for HCT116; RPMI 1640 (Sigma Chemical Co., St. Louis, MO) for HT29; and MEM Eagle (Life Technologies, Inc.) for HeLa; each was supplemented with 0.5 or 10% fetal bovine serum (Cansera, Canada) and 1% antibiotic/antimycotic solution (Sigma Chemical Co.). Cells were maintained at 37°C in an atmosphere of humidified air with 5% CO2 for LoVo, HT29, and HCT116, and without CO2 for SW480. Cancerous colonic tissues and corresponding noncancerous mucosae were excised from 24 patients during surgery, after informed consent had been obtained.

cDNA Microarray.
Fabrication of the cDNA microarray slides and construction of recombinant adenovirus have been described elsewhere (12 , 19) . For each analysis of expression profiles, we prepared two sets of slides containing duplicate sets of 9216 cDNA spots, to reduce experimental fluctuation. LoVo cells were infected at multiplicities of infection of 100 with adenovirus constructs expressing either wild-type AXIN1 (Ad-Axin) or LacZ (Ad-LacZ), a control gene. Total RNAs were extracted 72 h after infection, and T7-based RNA amplification was carried out using mRNA purified from the extracts as described elsewhere (19) . Five-µg aliquots of amplified RNA (aRNA) from LoVo cells with Ad-Axin or Ad-LacZ were labeled with Cy5-dCTP or Cy3-dCTP (Amersham Pharmacia Biotech, United Kingdom), respectively. Hybridization, washing, and detection were carried out as described previously (19) . Genes were excluded from further investigation when the intensities of both Cy3 and Cy5 were below 100,000 fluorescence units, and of the remainder, we selected for further evaluation those with Cy3/Cy5 signal ratios >2.0.

Semiquantitative RT-PCR Analysis.
Total RNA was extracted from cultured cells and clinical tissues using TRIZOL reagent (Life Technologies, Inc.) according to the manufacturer’s protocol. Extracted RNA was treated with DNaseI (Boehringer Mannheim, Mannheim, Germany) and reversely transcribed for single-stranded cDNAs using oligo(dT)12–18 primer with Superscript II reverse transcriptase (Life Technologies, Inc.). We prepared appropriate dilutions of each single-stranded cDNA for subsequent PCR amplification by monitoring the GAPDH gene as a quantitative control. Primer sequences were 5'-ACAACAGCCTCAAGATCATCAG-3' and 5'-GGTCCACCACTGACACGTTG-3' for GAPDH, and 5'-TGGCCATGGAGGA ACTCATC-3' and 5'-TGGGGAGCTTGTCATGACTG-3' for ENC1. All of the reactions involved initial denaturation at 94°C for 2 min followed by 18 cycles (for GAPDH) or 25 cycles (for ENC1) at 94°C for 30 s, 57°C for 30 s, and 72°C for 45 s, on a GeneAmp PCR system 9700 (PE Applied Biosystems, Foster, CA).

Western Blotting.
Western blotting with mouse anti-ß-catenin antibody (Transduction Laboratories, Lexington, KY) was performed as described elsewhere (20) .

Promoter Assay.
A transcriptional initiation site (TIS) of ENC1 was determined by a comparison of a human genomic sequence (GenBank accession no. NT-006596) and a cDNA sequence of ENC1 (GenBank accession no. NM-003633). To examine promoter activity of ENC1, we amplified five fragments, each corresponding to a part of the 5' flanking region of ENC1 by PCR, and cloned each of the products into an appropriate enzyme site of pGL3-Basic vector (Promega, Madison, WI). An activated form of ß-catenin (mut ß-catenin) was prepared by RT-PCR using a set of primers, 5'-AAGGATCCGCGTGGACAATGGCTACTCAAG-3' and 5'-GGACTCGAGACAGGTCAGTATCAAACCAGGCCAG-3', and RNA extracted from HCT116 colon cancer cells as a template, and cloned into an appropriate cloning site of pcDNA3.1 plasmid vector (Invitrogen). Mammalian expression plasmids of wild-type or dominant-negative form of Tcf-4 (wtTcf-4, dnTcf-4) was also prepared by RT-PCR using sets of primers as follows: TcfF1, 5'-AAGAATTCTGCTGGTGGGTGAAAAAAAAATGC-3', and TcfR1, 5'-CTACTCGAGTTCTAAAGACTTGGTGACGAGCGAC-3'; and TcfF3, 5'- AGGAATTCGTGCATCATGGTCCCACCACATCATAC-3', and TcfR1, respectively, and cloned into the pcDNA3.1 plasmid vector. Two µg each of the reporter plasmids and 1 µg each of the expression constructs were cotransfected with 0.5 µg of pRL-TK plasmid (Promega, Madison, WI) into HeLa cells using FUGENE6 (Boehringer Mannheim, Mannheim, Germany) to normalize the efficiency of transfection. Reporter assay was carried out using a dual-luciferase reporter assay system according to the supplier’s recommendations (Promega).

Growth Analysis.
The entire coding region of human ENC1 was cloned into expression vector pcDNA 3.1(+) (Invitrogen, Carlsbad, CA), under control of the cytomegalovirus promoter/enhancer. HCT116 colon cancer cells that expressed a high amount of ENC1 transcript (HCT116-ENC1 cells) were selected in medium containing 800 µg/ml geneticin. As a control, cells transfected with empty vector (HCT116-vector cells) were subcloned as well. HCT116-ENC1 and HCT116-vector cells were seeded at 2 x 105 cells/6 cm dish. Living cells detected by the trypan blue-exclusion method were counted in triplicate from days 0 to 7.

ELISA of CEA and Measurement of ALP Activity.
HT-29 colon cancer cells stably overexpressing wild-type ENC1 (HT29-ENC1 cells) were established. After being grown to ~50% confluence, HT29-ENC1 cells and controls (HT29-vector cells) were either treated with 2 mM NaB (Sigma Chemical Co.) or left untreated for 72 h, harvested, and lysed with O’Farrell lysis buffer as described previously (21) . The amount of CEA was analyzed by an ELISA using an Enzymun test kit (Boehringer Mannheim). ALP activity was measured as described elsewhere (22) .

Statistical Analysis.
The data were subjected to ANOVA and Scheffé’s F test.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Identification of ENC1 as a Gene Regulated by Axin.
We previously reported that wild-type AXIN1 was able to induce apoptosis in several colon cancer and liver cancer cell lines (12) . Because LoVo, a colon cancer line, was one of the most sensitive of these lines to the induction of apoptosis by AXIN1, we chose to use LoVo cells to search for genes regulated by AXIN1. Comparison of expression profiles of 9216 genes in LoVo cells infected with Ad-Axin or Ad-LacZ identified a number of genes expression of which was altered by Axin. Because transduction of AXIN1 reduces the level of ß-catenin in the nucleus and subsequently represses signaling mediated by the ß-catenin/Tcf complex (12) , we focused further on genes that appeared to be down-regulated by Axin. Among them, we confirmed down-regulation of ENC1 by semiquantitative RT-PCR experiments. We then examined expression of ENC1 in response to infection with Ad-APC, a construct that expresses the 20-amino acid repeat domain of APC, because this domain is able to down-regulate transcriptional activity of the ß-catenin/Tcf complex (23) . As expected, we observed decreased expression of ENC1 after infection with Ad-APC, and assumed that this reduction was a likely consequence of decreased activity of ß-catenin/Tcf (Fig. 1A)Citation .



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  		Fig. 1. A, Decreased expression of ENC1 in LoVo cells in response to either Ad-APC or Ad-Axin. RNAs and protein extracts were isolated from LoVo cells that were infected with the indicated adenoviruses at multiplicities of infection of 100 and incubated for 72 h. B, putative Tcf4-binding elements in the 5' flanking region and schematic presentation of various reporter plasmids of ENC1. The nucleotide positions from the putative transcription-initiating site were indicated with plus or minus number. C, reporter plasmids were cotransfected with various combinations of expression plasmids of pcDNA-mock, pcDNA-mut ß-catenin, pcDNA-wtTcf-4, and pcDNA-dnTcf-4 in HeLa cells. Reporter assay was carried out in triplicate at 48 h after transfection. Bars, SD. (*, Scheffé’s F test, P < 0.05).

 
Promoter Activity of ENC1 Is Up-Regulated by Introduction of ß-Catenin and Wild-Type Tcf4.
To test whether the promoter activity of ENC1 was regulated by ß-catenin/Tcf4 complex, we transfected the reporter plasmid P1 containing an ~2-kb fragment of the 5' flanking region of ENC1 with/without an activated form of mutant ß-catenin and wild-type Tcf4 into HeLa cells (Fig. 1B)Citation . The reporter activity of plasmid P1 was significantly enhanced by introduction of the activated form of ß-catenin and wild-type Tcf4 (Fig. 1C)Citation . Interestingly, the enhanced activity was reduced when P1 was cotransfected with the dominant-negative form of Tcf4, which suggested that Tcf4 affected the promoter activity of ENC1. To determine an element(s) responsible for the promoter activity, we further compared the promoter activity for each of the various deletion mutants of P1. The activity of P2 containing three putative Tcf-binding motifs (WWCAAAG) was higher than that of P5 (Fig. 1C)Citation . These data imply that a region encompassing -1654 and -745 is involved in the ENC1 promoter activity associated with the ß-catenin/Tcf4 complex. Although additional studies are necessary to clarify responsible element(s) in the promoter region, multiple elements are likely to be involved in the activation of ENC1 by ß-catenin/Tcf4 complex.

Expression of ENC1 in Colon Cancer Tissues.
Because accumulation of ß-catenin is a frequent feature of colorectal tumors, we examined expression of ENC1 in colon cancer samples and corresponding noncancerous tissues using semiquantitative RT-PCR, and detected increased expression in 17 (70.8%) of the 24 tumors examined (Fig. 2A)Citation . This result was consistent with the fact that ENC1 is up-regulated in response to activation of the ß-catenin/Tcf transcriptional complex.



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  		Fig. 2. A, expression of ENC1 in 12 colon cancer tissues (T) and corresponding noncancerous mucosae (N) as measured by semiquantitative RT-PCR. B, expression of ENC1 in 11 colon cancer cell lines as measured by semiquantitative RT-PCR. Expression of GAPDH served as the internal control in both experiments.

 
Growth Advantage Conferred by Overexpression of ENC1 in HCT116 Cells.
To disclose a potential role of ENC1 in colorectal tumorigenesis, we established HCT116-ENC1 cells that stably overexpressed ENC1 by transfecting an expression construct of pcDNA3.1-ENC1 into HCT116 colon cancer cells. The HCT116 line had shown the lowest expression of ENC1 among 11 colon cancer cell lines examined (Fig. 2B)Citation . When HCT116-ENC1 cells were cultured in medium containing 10% serum, they appeared to grow faster than the control cells, although the difference was not statistically significant (data not shown). However, when cultured in medium containing only 0.5% serum, HCT116-ENC1 cells proliferated at a markedly increased rate compared with the controls (Fig. 3A)Citation , indicating that ENC1-expressing HCT116 cells possessed a growth advantage under serum-depleted conditions. Furthermore, whereas HCT116-vector cells showed an apparent morphological change, i.e., foot-process formation, when grown in 0.5% serum, the morphology of HCT116-ENC1 cells did not change (Fig. 3, B and C)Citation .



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  		Fig. 3. A, growth rate of HCT116-ENC1 cells and control cells (HCT116-vector) cultured in medium containing 0.5% serum. This experiment was carried out in triplicate. Bars, SD. B, morphology of HCT116-ENC1 cells and HCT116-vector cells in serum-depleted medium.

 
Effect of ENC1 on Colonic Differentiation Induced by NaB.
Because ENC1 is involved in cell differentiation of neuronal and preadipocyte cells, we investigated whether ENC1 might also affect differentiation of colonic epithelial cells. On treatment with a differentiation-inducing agent, NaB, expression of ENC1 was elevated in HT29 colon cancer cells, which was correlated to the increase of two differentiation markers of the colon, ALP activity and CEA (24 , 25 ; Fig. 4ACitation ). To test whether NaB regulates expression of ENC1 through the ß-catenin/Tcf4 pathway, we examined expression levels of ß-catenin in response to treatment of NaB in the cells. Western blotting analysis using samples at various time points demonstrated unchanged expression of ß-catenin by NaB, which suggested that NaB might induce ENC1 in ß-catenin/Tcf4-independent manners (data not shown). Interestingly, overexpression of ENC1 reduced CEA in HT29 cells, whereas it did not affect ALP activity (Fig. 4, B and C)Citation . Furthermore, treatment of HT29-ENC1 cells, which stably overexpressed ENC1, with NaB, suppressed induction of both ALP activity and CEA; these two markers were elevated in HT29-vector cells ~4.5-fold and 2.2-fold, respectively (Fig. 4, B and C)Citation . These results implied that expression of ENC1 was up-regulated during differentiation and that its overexpression would suppress colonic differentiation.



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  		Fig. 4. A, ALP activity, CEA and expression of ENC1 in HT29 cells treated with NaB for indicated hours. Expression of ENC1 was analyzed by semiquantitative RT-PCR. B and C, effect of ENC1 on the activity of ALP (B) and CEA (C). Cells overexpressing ENC1 (HT29-ENC1) and control cells (HT29-vector) were treated (+) with NaB, or untreated (-), for 72 h. Measurements were carried out in triplicate. Bars, SD. (*, Scheffé F test, P < 0.01.)

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
The data presented here demonstrate that expression of ENC1 correlates well with the transcriptional activity of the ß-catenin/Tcf4 complex, because not only is its expression down-regulated by transduction of exogenous AXIN1 or APC into colon cancer cells but also the promoter activity is up-regulated by activated form of ß-catenin and wild-type Tcf4. Moreover, ENC1 expression was frequently elevated in the majority of primary colon cancer tissues that we examined. Our data suggest further that ENC1 may be involved in differentiation of colonic epithelial cells as well as in cell proliferation in environments in which nutrients and growth factors are limited.

A gene termed Pig10 (p53-induced protein 10), which has turned out to be identical to ENC1, was induced by overexpression of wild-type p53 in DLD1 colon cancer cells (26) . Because, in our experiments, we used a different colon cancer cell line, LoVo, which carries wild-type and inducible p53, we compared the expression of p53 in response to Ad-Axin and Ad-LacZ by Western blotting and observed no significant difference in the level of p53 protein. Furthermore, we confirmed by semiquantitative RT-PCR that expression of MDM2, a downstream gene of p53, was unchanged (data not shown). Because Pig10 had been isolated by serial analysis of gene expression (SAGE), using adenovirus-mediated gene transfer of p53, which induced apoptosis in DLD1 cells, it may be an indirect downstream gene, or a p53 target associated with apoptosis. Mutation of the p53 gene itself, which conceivably reduces transcription of downstream genes, is a frequent feature among colon cancers. Hence, elevated expression of ENC1 in colon cancers appears to occur in a p53-independent fashion. It appears that expression of ENC1 may be regulated by the ß-catenin/Tcf complex and by p53 or p53-regulated factors as well.

Cell differentiation requires coordination of many different events. Among those events, the most prominent include changes in cell shape and formation of a regulated architecture, both of which are closely associated with organization of the actin cytoskeleton. A member of the ezrin-radixin-moesin (ERM) family, NBL4 (EPB41L4), is also up-regulated by the ß-catenin/Tcf complex (10) . NBL4 is a band-4.1 protein that links actin filaments. Although it remains to be determined whether either NBL4 or ENC1 is regulated by the complex directly, they may both play roles in morphogenesis during cell differentiation through reorganization of the actin cytoskeleton.

Notably, although expression of ENC1 is most abundant in fetal brain, this gene is also expressed in fetal kidney, lung, heart, and liver; this activity is reduced or diminished in adult organs (14) . Therefore, ENC1 may play a crucial role during differentiation in a variety of cell lineages. Consistent with this notion, expression of ENC1 increases dramatically not only in neuroblastoma cells responding to retinoic acid, a differentiation-inducing agent, but also in preadipocytes treated with the phosphodiesterase inhibitor methylisobutylxanthine (MIX; Ref. 14 , 17 ).

Our study has added a novel role of ENC1, that is, in differentiation of colonic epithelial cells. We have also proved that overexpression of ENC1 does not affect differentiation of HT29 cells under normal conditions but prevents their differentiation when NaB is added. That result is in striking contrast to a reported observation that suppressing endogenous expression of ENC1 by transfection with a stable antisense construct prevented differentiation of adipocytes (17) . These data tempt us to speculate that ENC1 is required for early differentiation of adipocytes and colonic epithelial cells, but its overexpression inhibits full differentiation. During adipocyte differentiation, enhanced expression of ENC1 precedes induction of other differentiation markers such as peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), CCAAT/enhancer-binding protein {alpha} (C/EBP{alpha}), and adipocyte fatty acid-binding protein (aFABP); further differentiation results in decreased expression of ENC1, although other markers remain elevated (17) . These observations, together with a recent report showing inhibition of adipocyte differentiation by Wnt signaling, is consistent with the above hypothesis (27) . Alternatively, the data presented here may imply that differentiation is regulated by a tissue-dependent cofactor, or that moderate expression levels of regulatory proteins are essential for differentiation. These possibilities remain to be examined to clarify the role of ENC1 in cellular differentiation.

Expression of ENC1 is up-regulated in various types of brain tumors (18) . Because we also found its enhanced expression in the majority of colon cancers that we examined, activation of ENC1 may be a feature of tumorigenic mechanisms common to colon and brain. Our hypothesis raises a possible scenario in which activated ENC1 may lead to neoplasms by preventing regulated differentiation of colonic mucosae and neural cells.

In conclusion, our discovery that ENC1 is a downstream target of the ß-catenin/Tcf4 complex brings new insight concerning colorectal tumorigenesis. Further investigation of the function of ENC1 should provide a more profound understanding of the mechanisms involved. In addition, inactivation of ENC1 may conceivably serve in the future as a novel therapeutic intervention for treatment of patients with colon cancer.


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

1 Supported in part by Research for the Future Program Grant 00L01402 from the Japan Society for the Promotion of Science. Back

2 To whom requests for reprints should be addressed, at Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5372; Fax: 81-3-5449-5433; E-mail: yusuke{at}ims.u-tokyo.ac.jp Back

3 The abbreviations used are: ENC1, ectodermal-neural cortex 1; APC, adenomatous polyposis coli; Tcf, T-cell factor; RT-PCR, reverse transcription-PCR; ALP, alkaline phosphatase; CEA, carcinoembryonic antigen; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; NaB, sodium butyrate; LEF, lymphocyte enhancer factor. Back

Received 4/ 3/01. Accepted 9/13/01.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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