Azoxymethane-induced pre-adipocyte factor 1 (Pref-1) functions as a differentiation inhibitor in colonic epithelial cells

Mei Dong1, Kishore Guda1, Prashant R. Nambiar1, Masako Nakanishi1, Alexander C. Lichtler2, Mitsuo Nishikawa3, Charles Giardina4 and Daniel W. Rosenberg1,5

1 The University of Connecticut Health Center, Center for Molecular Medicine, Farmington, Connecticut, USA, 2 The University of Connecticut Health Center, Department of Genetics and Developmental Biology, Farmington, Connecticut, USA, 3 Pharmaceutical Research Laboratories, Pharmaceutical Division, Kirin Brewery Company Limited, Tokyo, Japan and 4 The University of Connecticut, Department of Molecular and Cell Biology, Storrs, Connecticut, USA

5 To whom correspondence should be addressed Email: rosenberg{at}nso2.uchc.edu


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Inbred mice differ dramatically in their sensitivity to the colon carcinogen, azoxymethane (AOM). Identifying genes associated with this differential susceptibility in mice may ultimately reveal molecular mechanisms responsible for colon carcinogenesis. A cDNA array approach was taken to study gene expression changes induced by AOM in the colons of sensitive (A/J) and resistant (AKR) mice. Among the genes represented on the array, pre-adipocyte factor 1 (Pref-1), associated previously with suppression of adipocyte differentiation, was induced specifically by AOM in the distal colons of sensitive A/J mice (5.4-fold). Reverse transcription–PCR followed by sequence analysis revealed the presence of four alternative splice variants of Pref-1 mRNA in the colon. The potential significance of Pref-1 in colon tumorigenesis was explored in colon cancer cells infected with a retroviral construct containing the major splice variant. Over-expression of Pref-1A in HT-29 cells led to a marked resistance to butyrate-induced differentiation and growth inhibition. Our data indicate that Pref-1, a protein that suppresses differentiation and promotes colonocyte growth, may account in part for the sensitivity of A/J mice to AOM-induced carcinogenesis. In addition, detection of Pref-1 in a human colon tumor cell line suggests that it may also participate in human colon tumorigenesis.

Abbreviations: AOM, azoxymethane; BrdU, bromodeoxyuridine; EGF, epidermal growth factor; EGR1, early growth response factor 1; IIP-1, interferon inducible protein 1; IRF-1, interferon response factor 7; Pref-1, pre-adipocyte factor 1


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The organotropic carcinogen, azoxymethane (AOM), produces tumors and pre-neoplastic lesions within the distal colons of rodents. AOM-induced tumors share a number of pathological features with sporadic human colorectal cancer (14). The AOM rodent model thus provides a useful experimental system for studying the molecular and pathological changes associated with the human disease. Of particular relevance is the observation that inbred mice demonstrate profound differences in their relative sensitivities to AOM. For example, sensitive A/J mice develop as many as 40 tumors in the distal colon, whereas resistant AKR mice rarely develop any tumors (5,6). However, pre-neoplastic aberrant crypt foci (ACF) are formed in the colons of both strains. Although ACF begin to appear within 2–4 weeks of post-carcinogen exposure, the percentage of dysplastic ACF in the sensitive A/J colons are significantly higher than in the AKR strain. Thus, the identification of molecular alterations that distinguish differential susceptibility may lead to the identification of genes involved in the early stages of colon tumorigenesis.

In the present study, a gene profiling approach was used to compare global expression patterns in the distal colons of sensitive and resistant mice 1 week following carcinogen exposure. Of the 1176 genes represented on the cDNA array, pre-adipocyte factor 1 (Pref-1) was found to be differentially induced in the distal colons of the carcinogen-sensitive A/J mice. Pref-1, also referred to as delta-like protein, is a transmembrane protein with six epidermal growth factor (EGF)-like sequences within the extracellular domain, a single transmembrane domain and a short intracellular tail (7). Because of its overall structure and amino acid homology, Pref-1 belongs to the family of EGF-like homeotic proteins that are involved in regulating growth and differentiation through cell–cell interaction (8). Pref-1 has been demonstrated to act as an inhibitor during the differentiation process of pre-adipocytes to mature adipocytes (9). The signal transduction pathway mediated by Pref-1 is not well understood, but it has been hypothesized to exert its inhibitory effect on cell differentiation through an unidentified receptor via its EGF-like domains, either through a paracrine or juxtacrine mechanism (10).

In the present study, we have identified the mouse strain-specific induction of Pref-1 in response to exposure to AOM. Functional analysis of Pref-1 in human colonocytes demonstrates that Pref-1 influences colonocyte differentiation and proliferation and is expressed in a subset of human colon cancer cell lines. The data suggest that Pref-1 may play a role in AOM-induced colon tumorigenesis, and possibly human colon cancer development as well.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Treatment of animals and tissue sample preparation
Five week-old male A/J and AKR mice (Jackson Laboratories, Bar Harbor, ME) were housed in a temperature-controlled environment (23°C ± 1) with a 12 h light/dark cycle. Mice were provided with Purina laboratory rodent chow 5001 and water ad libitum. After an acclimation period of 1 week, mice of each strain were divided randomly into two equal groups (n = 5). One group was treated with AOM dissolved in saline by i.p. injection at a dose of 10 mg/kg body wt once per week for 6 weeks. The remaining group received saline alone and served as the vehicle control. Mice were killed 1 week after the last dose. The entire colon was removed and flushed with ice-cold PBS buffer. Colons were slit open longitudinally. The portions for RNA and protein were frozen immediately in liquid nitrogen and stored at –80°C. The remaining portion was fixed in 10% neutral-buffered formalin for 6 h and embedded in paraffin for subsequent histopathological examination and immunostaining.

Gene expression array analysis
The gene expression profiles were obtained using the Clontech Atlas mouse 1.2 K cDNA expression array (Clontech, Palo Alto, CA) consisting of cDNAs for 1176 genes. One week after the last dose, vehicle control and AOM-treated AKR and A/J mice were killed. Total RNA isolated from the distal colon of one animal from each group using Qiagen RNeasy midi kit was treated with DNase and used for cDNA probe synthesis in the presence of [32P]dATP. Labeled cDNA probes were purified on ChromaSpin-200 columns, and fractions corresponding to cDNA were pooled and used in the hybridization reaction, which was performed overnight at 68°C. Signals on the array were detected using a phosphorimager, and subsequent data analysis was performed using AtlasImage 1.5 software. Ratios ≥2- or ≤0.5-fold were considered to be significant.

Multiprobe RPA
Total cellular RNA was isolated from frozen vehicle control and AOM-treated distal colons using the Qiagen RNeasy kit (Qiagen, Valencia, CA) following the manufacturer's instructions. To make the anti-sense probe for each of the genes, a reverse transcription (RT)–PCR reaction was first carried out according to a standard protocol (11). The primers used in the PCR reactions were as follows: Pref-1, 5'-TCT GCG AAA TAG ACG TTC GGG CTT GC-3' and 5'-GCC ATC GTT CTC GCA TGG GTT AGG G-3'; interferon response factor 7 (IRF-7), 5'-CAC CGA GCG CAG CCT TGG GTT C-3' and 5'-AGA AGCGTC TCT GTG TAG TGC AGC-3'; early growth response factor 1 (EGR1), 5'-GTT CCT CGG TGC TGC CGG AAC CC-3' and 5'-GGA GGA TTG GTA ATG CTC ACG AGG C-3'; interferon inducible protein 1 (IIP-1), 5'-CAT GGG CGA GTG TCT GAA AGT GTA CC-3' and 5'-TCT AGG ATG TTC TTG GTG TCC TGG GC-3'; signal transducer and activator of transcription 1, 5'-TCA GCA AGG AGC GAG AAC GCG CTC-3' and 5'-CCA GTT CGC TTA GGG TCG TCA AGC-3'; HOXD13, 5'-TCG GCA CGA GGC ATA CAT CTC CAT-3' and 5'-CGC CGC CGC TTG TCC TTG TT-3'; and HPRT (loading control), 5'-GTA ATG ATC AGT CAA CGG GGG AC-3' and 5'-CCA GCA AGC TTG CAA CCT TAA CCA-3'. cDNA fragments of different length were cloned into the pGEM-T Easy vector (Promega, Madison, WI). The 32P-labeled anti-sense RNA probes were synthesized using the RiboQuant In vitro Transcription kit (PharMingen, San Jose, CA) according to the manufacturer's instructions. Five microgram RNA samples were incubated with the anti-sense probes overnight at 56°C and then digested by treatment with RNase. The protected double-stranded RNA fragments were resolved on 7% denaturing gels, which were then exposed to X-ray film at –70°C. Image densitometry was performed using NIH image software.

Immunostaining and fluorescence microscopy
Five micrometer formalin-fixed, paraffin-embedded colon tissue sections from AKR and A/J mice were incubated at room temperature for 1 h with goat polyclonal anti-Pref-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:2000 in 5% normal rabbit serum. Sections were washed in PBS and further incubated with Alexa Fluor 488-conjugated rabbit anti-goat immunoglobin G (IgG) (Molecular Probes, Eugene, OR) at room temperature for 30 min. After washing, the sections were counterstained with hematoxylin for 20 s and examined for fluorescence using a Zeiss 410 laser-scanning confocal microscope. As a negative control, duplicate sections were immunostained with goat IgG in place of the primary Pref-1 antibody.

Protein extraction and western analysis
Fresh tissue samples from AOM- and vehicle-treated mice were rinsed with cold HBSS–10 mM DTT and incubated with HBSS–10 mM EDTA on ice for 1 h with constant agitation. Dissociated colonocytes were collected by centrifugation at 5000 r.p.m. for 10 min, and resultant pellets were washed twice with cold PBS. The cells were then lysed by incubation in RIPA buffer containing protease inhibitor cocktail maintained on ice for 30 min. Samples were then centrifuged at 10 000 g for 15 min and the total protein content of the supernatant was quantified with the Bio-Rad DC Protein Assay (Bio-Rad Laboratories, Hercules, CA). Fifty micrograms of protein was separated on a 10% SDS–PAGE gel and then electrotransfered onto a nitrocellulose membrane. The membrane was probed with hamster anit-mouse-Pref-1 antibody at a concentration of 1 µg/ml (12). Goat-anti-hamster horseradish peroxidase-conjugated secondary antibody was used at a dilution of 1:5000 (Research Diagnostics, Flanders, NJ), and immunoreactivity was visualized using the ECL western blot analysis system (Santa Cruz Biotechnology, CA).

For extraction of cell membrane protein, cells were harvested and the cell pellet was re-suspended in hypotonic buffer (20 mM Tris, pH 7.4; 5 mM MgCl2; 5 mM CaCl2) and incubated on ice for 15 min. The cell suspension was then homogenized on ice followed by centrifugation at 1000 g for 10 min to remove cell nuclei. The supernatant was collected and further centrifuged at 18 400 r.p.m. for 2 h at 4°C. The resultant pellet containing the cell membrane fraction was dissolved in 0.5% SDS in Tris–EDTA (50 mM Tris, 1 mM EDTA, pH 8.0) at 95°C. Rabbit-anti-human Pref-1 antibody was obtained from Alpha Diagnostic (San Antonio, TX). Immunoblotting was carried out as described above.

For p21 western analysis, cell pellets were lysed by incubation with buffer A (cytoplasmic extract) at 4°C for 8 min followed by incubation with buffer C (nuclear extract) as described previously (13). A 40-µg aliquot of nuclear protein was resolved on a 4–15% SDS–PAGE gel and transferred to 0.2-µm PVDF membrane. p21 was detected using mouse monoclonal antibody p21WAF1 Ab-5 (NeoMarkers, Frement, CA) at a concentration of 2 µg/ml.

RT–PCR analysis
The RT reaction was carried out according to standard protocol (11). Primers flanking the alternative-splicing region of Pref-1 were used for PCR amplification, forward (1306–1328): 5'-TGC TTA GAT CTC CTC ATC ACC AG-3' and reverse (763–787): 5'-CCA GCT TTA TTC GTC GAC AAG ACC T-3'. The PCR products were separated on a 2% agarose gel. The individual bands were purified using Zymoclean Gel DNA Recovery kit (Zymo Research, Orange, CA), and cloned into pGEM-T Easy vector (Promega) and sequenced. For the RT–PCR of Pref-1 in cultured human colon tumor cell lines, the following primers were used: Pref-1, 5'-TGG ACG GTG GCC TCT ATG AAT GCT-3', and 5'-CGC GCT TCT TGA CAC AGG TGA GAC-3'; GAPDH, 5'-CCA TGG AGA AGG CTG GG-3' and 5'-GGT CAT CCA TGA CAA CTT TG-3'.

Retroviral-vector construction and cell infection
Full-length Pref-1 cDNAs were PCR-amplified using the following primers: forward, GCG AAT TCA TGA TCG CGA CCG GAG C, and reverse, GCG GAT CCG CTG TCA ATC TTC TCG GGG AAG. PCR products were resolved on a 2% agarose gel. Pref-1A was purified from the gel and cloned into the EcoR1 and Not1 site of replication-defective, Maloney murine sarcoma virus-based retroviral vector, pMg1-IRES-EGFP (Figure 7A). This vector was derived from Cux-1 retroviral vector (14) by replacing the Cux-1 cDNA with a polylinker. This construct was then stably transfected, according to the LipofectAMINE 2000 protocol (GIBCO), into the 293GPG packaging cell line, which expresses the structural genes gag and pol that are necessary for retroviral vector assembly. Furthermore, the 293GPG packaging cell line also expresses the G glycoprotein of vesicular stomatitis virus (VSV) under the control of the tetracycline-responsive element (tet-off system). The VSV G protein can markedly enhance the infectivity of the retroviral vector through interaction with the phospholipids of the cell membrane. After transfection, cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS, 0.5 U/ml Pen-Strep, 0.1 µg/ml tetracycline, 2 µg/ml puromycin and 0.3 mg/ml G418 for 24 h. Tetracycline was then removed from the medium to enable production of infective virus. Virus-containing medium was collected every 24 h for 3–4 days, filtered through a 0.2-µm filter and immediately stored at –80°C. For infection, filtered media was supplemented with polybrene to a final concentration of 8 µg/ml, and added to cultured HT-29 cells. After an 8-h incubation, dishes were washed briefly with PBS, then fresh DMEM with 10% FBS was added. Finally, GFP-positive cells were sorted by flow cytometry. Empty retroviral vector was used in parallel as a control.



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Fig. 7. (A) RT–PCR analysis of Pref-1 expression in human colon tumor cell lines. Note the presence of Pref-1 mRNA in HCT-116, but not in Caco-2, DLD1 and HT-29 cells. (B) Full-length Pref-1A cDNA was PCR amplified and cloned into the retroviral expression vector, pMg1-IRES-EGFP. (C) Cultured HT-29 cells were infected with the retroviral vector and expression of Pref-1 was detected by western blot analysis of proteins extracted from the cell membrane. Note that Pref-1 protein was detected only in cells infected with vectors carrying Pref-1 cDNA, but not with mock vectors.

 
Butyrate treatment and alkaline phosphatase and caspase 3 activity determinations
For the alkaline phosphatase (ALP) assay, equal amounts of cells were seeded in a 12-well plate and treated with various concentrations of butyrate for 24 h after they reached 80% confluency. The cells were collected, washed in ice-cold PBS and lysed by sonication in RIPA buffer, pH 9.7. Protein concentration was measured using the Bio-Rad DC Protein Assay. Fifty micrograms of protein, at a concentration of 2.5 µg/µl, was incubated with 200 µl ALP substrate (Sigma) in a 96-well plate at 37°C for 30 min. Optical density was measured at 405 nm in a Bio-Rad microplate reader. For the caspase-3 activity, equal numbers of cells were lysed and centrifuged at 10 000 g for 10 min at 4°C. The supernatant was then incubated with the colorimetric caspase-3 substrate I (Calbiochem) for 10 min at 37°C. Absorbance at 405 nm was recorded every 5 min for 2 h in a microplate reader.

Bromodeoxyuridine cell proliferation assay
The bromodeoxyuridine (BrdU) proliferation assay was performed according to the manufacturer's protocol (Oncogene Research Products, San Diego, CA). Briefly, 80% confluent cells growing in 100 µl culture medium in 96-well plates were treated with varying concentrations of butyrate for 20 h. Twenty microliters of BrdU label (1:2000 dilution of the stock solution) were added to the medium and the cells were further incubated for 4 h. Cells were fixed and probed with anti-BrdU antibody (1:100) for 1 h. The plate was washed and incubated with peroxidase-conjugated secondary antibody for 30 min. Finally, 100 µl of substrate solution was added to each well and absorbance was read at dual wavelengths of 450 and 595 nm using a microplate reader.

Statistical analysis
The Generalized linear model using SAS software was used for data analysis. Significant differences were determined by the probability of difference (PDIFF) between the means. A P-value <0.05 is considered statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Strain-specific induction of Pref-1 following AOM treatment
To compare gene expression profiles in the colons of carcinogen-exposed mice, RNA was isolated from distal colons 1 week after the last injection of AOM and subjected to microarray analysis. The Atlas mouse 1.2 cDNA expression microarray, which consists of cDNAs for 1176 genes belonging to a wide range of gene families, was used for this analysis. Expression of the entire gene set was normalized by comparing the expression of several housekeeping genes on the arrays. Genes exhibiting significant alterations are shown in Figure 1. Further confirmation was performed by quantitative multi-probe RPA analysis using additional RNA samples isolated from the same experimental group. As shown in Figure 2, RPA results demonstrated similar expression alterations in the colons of carcinogen-treated animals. IIP-1, IRF-7 and STAT1 were increased by AOM to a comparable extent within the colonic epithelium of AKR and A/J mice (2.70-, 3.66- and 2.74- versus 2.85-, 3.35- and 3.33-fold, respectively), while EGR1 expression was significantly decreased in both strains (0.36- versus 0.37-fold). Only Pref-1 was found to be differentially expressed upon carcinogen exposure. As shown in Figures 1 and 3A, Pref-1 mRNA was increased 5.4-fold in the colons of A/J mice in response to AOM. No colonic expression of Pref-1 was found in similarly treated, AOM-resistant AKR mice. This finding was confirmed by western blot analysis (Figure 3B), and by the presence of intense apical membrane staining of Pref-1 using immunofluorescence (Figure 4). Since the primary goal of our study was to identify differentially regulated genes in carcinogen-exposed colons of sensitive and resistant mice, further functional studies were focused on Pref-1.



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Fig. 1. Comparison of gene expression changes in the colons of AKR and A/J mice following AOM exposure. Gene expression alterations induced by AOM in the distal colons of AKR (A) and A/J mice (B) 1 week after the last treatment. Expression of the entire gene set was normalized by comparing the expression of several house keeping genes on the array. Only those genes exhibiting at least a 2-fold difference in expression between the AOM treatment groups compared with the vehicle-treated controls were considered to be significantly altered and are presented.

 


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Fig. 2. Multi-probe RPA validation of microarray data. RNA extracted from AKR and A/J control (n = 3 and 4) and AOM-treated (n = 3 and 4) mice were used. Quantitative analysis of mRNA levels was performed using NIH image analysis software. Statistical analysis was performed using the Generalized linear model procedure followed by PDIFF post-hoc analysis for comparing the means. Note the significant up-regulation of Stat1, IRF-7 and IIP-1 and down-regulation of EGR1 in both the sensitive and resistant strains. HPRT was used as a loading control for normalization.

 


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Fig. 3. Strain-specific induction of Pref-1 following AOM treatment. (A) Pref-1 mRNA levels in normal and AOM-exposed distal colons of AKR and A/J mice were determined by RPA. HPRT was used as a loading control for normalization. Note the increase of Pref-1 mRNA (5.4-fold, P < 0.01) only in the AOM-exposed A/J mouse colon. (B) Comparison of Pref-1 protein levels in the distal colons of A/J mice with (n = 4) or without AOM treatment (n = 4). Note the presence of Pref-1 protein only after exposure to AOM.

 


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Fig. 4. Immunofluorescence analysis of Pref-1. Note the presence of intense apical membrane staining of Pref-1 in the AOM-exposed A/J colon.

 
Tissue-specific induction of Pref-1 and alternative splicing of Pref-1 mRNA
AOM undergoes biotransformation in the liver to methylazoxymethanol (MAM), the reactive and carcinogenic metabolite. MAM is widely distributed via the systemic circulation (15). However, AOM produces tumors that are restricted almost exclusively to the distal colon, suggesting that genetic alterations specific to the colon may promote a tumorigenic response. To determine whether Pref-1 induction was also restricted to the distal colon, we examined Pref-1 expression by RPA in a panel of tissues isolated from AOM- or vehicle-exposed mice. As shown in Figure 5, Pref-1 expression was significantly elevated (6.2-fold) in the adipose tissue of AOM-exposed A/J mice. However, within the gastrointestinal tract, Pref-1 induction (5.7-fold) was restricted to the distal colons of A/J mice. No Pref-1 transcripts were detected by RPA in either the proximal colon or small intestine. In addition, Pref-1 induction was not observed in the adipose tissue of AKR mice (data not shown).



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Fig. 5. Tissue-specific induction of Pref-1 in A/J mice. RPA analysis of Pref-1 mRNA levels was performed in multiple tissues dissected from control and AOM-treated mice. Data represent one of three independent experiments. Note the marked induction of Pref-1 in adipose tissue and distal colon, but not in the other organs.

 
It has been shown that Pref-1 pre-mRNA undergoes alternative splicing in pre-adipocytes (16). Of the four splicing products, only Pref-1A and Pref-1B are biologically active (17). To determine the splicing profile of Pref-1 in the colon following carcinogen exposure, RT–PCR was performed using primers flanking the alternative-splicing region. As shown in Figure 6, a total of four PCR products were detected, which were confirmed by sequence analysis to represent alternative-splicing variants. Our data also revealed a similar profile of Pref-1 splice variants in the colon as reported previously in pre-adipocytes (16). In addition, sequence analysis of the full-length cDNA of the most abundant isoform, Pref-1A, revealed no mutations (data not shown).



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Fig. 6. Alternative splicing of Pref-1 mRNA. Primers flanking the alternative splicing region of Pref-1 mRNA were used for RT–PCR reaction. A total of four PCR products were detected and confirmed to be alternative splicing products by sequence analysis. Note Pref-1A is the most abundant isoform.

 
Resistance of the HT-29 cell line over-expressing Pref-1A to butyrate induced growth arrest and differentiation
As mentioned previously, Pref-1 is considered to play a critical role in maintaining the undifferentiated phenotype of pre-adipocytes (7). It has also been detected in a variety of human tumor cell lines and tumors with neuroendocrine features (7). However, the status of Pref-1 in human colon cells has not been previously reported. Therefore, in the present study, we screened a panel of human colon cancer cell lines and found that Pref-1 was expressed in HCT-116, but not in HT-29, CaCo2 or DLD1 cell line (Figure 7B). To establish a functional role for Pref-1 in colonocytes, Pref-1A, which represents the most abundant isoform in the mouse, was cloned into a retroviral expression vector (Figure 7A) and subsequently infected into HT-29 cells that do not naturally express Pref-1. Expression of the gene was confirmed by western blot analysis of proteins extracted from the cell membrane (Figure 7C).

Since Pref-1 has been shown to inhibit the differentiation of pre-adipocytes, we examined whether the introduction of the retroviral vector containing Pref-1 may produce a comparable effect in HT-29 cells. To test this possibility, HT-29 cells were exposed to butyrate, a short-chain fatty acid present within the colonic lumen that is known to induce differentiation and cell cycle arrest in colonic epithelial cells at a physiological concentration (18,19). A significant reduction in the cellular response to butyrate was observed; the butyrate-induced increase in alkaline phosphatase activity (a differentiation marker) and decrease in BrdU incorporation (a proliferation marker) was suppressed in Pref-1-expressing cells relative to control cells (Figure 8A and B). It has been demonstrated that growth arrest mediated by butyrate is due mainly to the induction of p21WAF1 (20). In the present study, we also observed a dose-related increase in p21 levels in control HT-29 cells following butyrate treatment. However, in cells over-expressing Pref-1, the butyrate-induced increase in p21 levels was absent (Figure 8C). Furthermore, a reduced apoptotic response to butyrate treatment, as measured by reduced caspase 3 activity was also observed in Pref-1 over-expressing cells (Figure 8D). Taken together, the in vitro functional studies clearly demonstrate that Pref-1 over-expression in HT-29 cells confers resistance to butyrate-induced differentiation, growth inhibition and apoptosis.



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Fig. 8. Effects of Pref-1 expression on the response of HT-29 cells to butyrate. (A) Cells were treated with various concentrations of butyrate for 24 h, alkaline phosphatase activity, as indicated by the absorbance at 405 nm, was examined as a marker for differentiation. (B) Cells were first treated with various concentrations of butyrate for 20 h, and then co-incubated with BrdU label for 4 h. BrdU incorporation, a marker for cell proliferation, was then measured using BrdU antibody. Each data point represents the mean ± SD of three individual measurements. (C) Nuclear proteins were extracted and p21 level was measured by western blot analysis. Note the absence of p21 induction in cells over-expressing Pref-1. Data were presented as mean ± SD of three individual experiments. (D) After 24 h treatment with butyrate, cells were collected and the cell lysate was incubated with a colorimetric caspase-3 substrate. Caspase 3 activities were measured every 5 min for 2 h and presented as absorbance at 405 nm. This experiment was performed three times in triplicate with similar results.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
As in human populations, the genetic background of inbred mice plays an important role in conferring differential susceptibility to colon carcinogenesis (6). Identifying genes associated with this differential sensitivity will improve our understanding of the molecular mechanisms underlying colon tumorigenesis. In the present study, we employed a cDNA array approach to study gene expression patterns at an early time point in the colons of sensitive (A/J) and resistant (AKR) mice in response to AOM treatment. Of the 1176 genes analyzed, IRF-7, Stat-1, IIP-1 and EGR1 were found to be similarly altered in both AKR and A/J mice after carcinogen exposure. IRF-7 is a transcription factor involved in the induction of interferon genes in response to viral infection and DNA-damaging agents (21,22). Stat-1 is a transcription factor and a downstream effector of the IFN-{gamma} signaling pathway. This pathway has been demonstrated to play an essential protective role against chemically induced or spontaneous tumors (23,24). IIPs are a family of chemokines that are released upon IFN-{gamma} signaling and serve as chemoattractants for CD4-positive lymphocytes (23). Overall, our data with respect to IRF-7, Stat-1 and IIP-1 suggest that an interferon-mediated, anti-tumorigenic immune surveillance system has been activated in response to AOM treatment. Expression of the EGR1 gene was found to be down-regulated by AOM in both strains. EGR1 is an immediate early gene product that has been implicated in multiple cellular processes including growth, differentiation and apoptosis. It has been found to be decreased or absent from small lung carcinomas, breast cancers and colon cancers, as well as in human gliomas, providing the rationale for its potential role as a tumor suppressor (25). The functional significance of down-regulation of EGR1 in the colons of carcinogen-exposed mice remains to be determined.

The expression patterns noted above, however, do not provide discrimination between the sensitive and resistant colons. Of the panel of tumor-related genes that were screened on the cDNA microarray, only Pref-1 was found to afford discrimination between A/J and AKR colons. This finding was rather unexpected since previous studies of Pref-1 have been focused almost exclusively on its role as an inhibitor of pre-adipocyte differentiation (10). However, the role of Pref-1 in pre-adipocytes may provide clues into its functional significance within the colonic epithelium. The expression of Pref-1, which is high in pre-adipocytes, is essentially abolished during differentiation of pre-adipocytes into mature adipocytes (16). It has been further demonstrated that down-regulation of Pref-1 is necessary for cellular differentiation to occur, a conclusion that is based on the observation that constitutive expression of Pref-1 inhibits the conversion (9). Despite its fundamental role in the regulation of adipocyte differentiation, there is relatively little information regarding its regulation at the genetic level. Therefore, to learn more about the strain-specific induction of Pref-1, we sequenced the 1.1-kb promoter region of Pref-1 in both AKR and A/J mice. Sequence analysis revealed no polymorphic variation across this region (data not shown). The similarity of promoter sequences suggests that strain-specific regulation of Pref-1 expression may be due to difference(s) in upstream transcription regulatory events that are specifically activated within the A/J mice. Interestingly, sequence analysis of the Pref-1 promoter did reveal three potential response elements for the ß-catenin/TCF transcription factor, which has been proposed to play an important role in AOM-induced colon tumorigenesis (26,27). Further studies are underway to determine whether Pref-1 is indeed a transcriptional target of ß-catenin, and whether there exist strain-specific activation pathways that would confer such a differential response.

To gain additional insight into the potential role of Pref-1 in regulating colonocyte differentiation and turnover, the effects of Pref-1 on differentiation were evaluated in HT-29 colon tumor cells. Butyrate, a fatty acid that is endogenous to the microenvironment of the colon, has been proposed to be an important regulator of cell differentiation (19). In the present study, we found that the introduction of Pref-1 into HT-29 cells using a retroviral expression system attenuated their response to butyrate-induced differentiation and growth arrest. These in vitro observations suggest that the induced expression of Pref-1 by carcinogen exposure in A/J mice may result in increased resistance of colonocytes to butyrate, as well as other luminal factors that may promote growth arrest and differentiation. Such a response could potentially promote the malignant conversion of colonocytes by facilitating the proliferation of genetically damaged cells that, in the absence of Pref-1, would otherwise be targeted for programmed cell death.

Apart from cultured pre-adipocytes, Pref-1 is widely expressed in mouse embryonic tissues. However, in adult mouse, its expression is confined to the adrenal gland, anterior pituitary, endocrine pancreas and placenta (7,28). It has also been reported to be expressed in human tumors with neuroendocrine features (7) and in human uterine leiomyomas (29). In the present study, we also detected Pref-1 mRNA in HCT-116 cells, one of the four human colon tumor cell lines that were investigated. Considering the ability of Pref-1 to inhibit butyrate-induced growth arrest and differentiation of colonocytes, our findings suggest that Pref-1 may play a role in mediating carcinogenesis within a subset of human colorectal cancers as well.


    Acknowledgments
 
We thank Mary Louise Stover for her excellent technical advice on the retroviral vector system. We also acknowledge the support of the UCONN Health Center Core for Musculoskeletal and Skin Diseases. These studies were supported by NIH grant CA 81428-01 to D.W.R.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 28, 2003; revised July 10, 2004; accepted July 18, 2004.





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