Role of gut-enriched Krüppel-like factor in colonic cell growth and differentiation

Jue-Lon Shie1, Zhi Y. Chen1, Michael J. O'Brien2, Richard G. Pestell3, Mu-En Lee4, and Chi-Chuan Tseng1

1 Section of Gastroenterology, 2 Department of Anatomic Pathology, Boston Veterans Affairs Medical Center and Boston University School of Medicine, Boston 02118; 4 Cardiovascular Biology Laboratory, School of Public Health, Harvard University, Boston, Massachusetts 02115; and 3 Department of Development and Molecular Biology and Medicine, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Cancer cells differ from normal cells in many aspects, including hyperproliferation and loss of differentiation. Recent research has focused on the role of transcription factors in regulating abnormal cell growth. Gut-enriched Krüppel-like factor (GKLF) is a newly identified eukaryotic zinc finger protein expressed extensively in the gastrointestinal tract. In the current study, we demonstrated that GKLF mRNA levels were significantly decreased in the dysplastic epithelium of the colon, including adenomatous polyp and cancer. GKLF immunostains in the normal colon were higher at the surface epithelium and gradually decreased toward the crypt, but this gradient was not present in the adenomatous and cancerous mucosa. Constitutive overexpression of GKLF DNA in a human colonic adenocarcinoma cell line (HT-29) decreased [3H]thymidine incorporation, whereas suppression of GKLF gene increased DNA synthesis, indicating that downregulation of the GKLF gene might contribute to cellular hyperproliferation. Cyclin D1 (CD1) protein level and CD1-associated kinase activity were decreased in HT-29 cell overexpressed GKLF cDNA, and CD1 promoter activity was profoundly suppressed by GKLF. When HT-29 cells were cultured in the presence of sodium butyrate, GKLF mRNA levels increased as cells acquired more differentiated phenotypes. These results suggest that GKLF plays an important role in regulating cell growth and differentiation in the colonic epithelium and that downregulation of GKLF expression may cause colonic cells to become hyperproliferative. Furthermore, GKLF appears to be a transcriptional repressor of the CD1 gene.

colon cancer; adenoma; proliferation; transcription factor


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE COLONIC EPITHELIUM UNDERGOES rapid turnover in which cell production is usually balanced by cell extrusion. Within a period of 3-8 days, colonic epithelial cells derived from a stem cell population at the base of the crypt migrate from a region of proliferation in the basal three-quarters of the crypt to the surface and assume a more differentiated phenotype (17). Apoptotic cells are seen among the terminally differentiated cells near the surface epithelium and the top of the crypt (8). During malignant transformation of colonic mucosa, the region of proliferation expands and differentiation is abnormal or disrupted (17). The current concept of colonic carcinogenesis begins with damage to normal cellular genes, the activation of oncogenes, and the loss of tumor suppressor genes (25). A series of genetic events has been observed in the progression from normal to hyperproliferative epithelium, adenoma, carcinoma, and invasive carcinoma of the colon. It has been postulated that increases in intestinal cellular proliferation might lead to gene mutation and, eventually, carcinogenesis. Alternatively, spontaneous mutation at the cellular level increases cellular proliferation, resulting in clonal expansion. Although many genetic alterations in colonic carcinogenesis have been elucidated, the molecular events that regulate cellular hyperproliferation have not been fully examined.

Gut-enriched Krüppel-like factor (GKLF) is a newly identified eukaryotic zinc finger protein (7, 21) that has three carboxy-terminal zinc fingers with a high degree of homology to the tissue-specific transcription factors lung Krüppel-like factor, erythroid Krüppel-like factor (EKLF), and BTEB2 (1, 5, 18). The amino terminus of GKLF possesses multiple proline and serine residues consisting of transcriptional activation domains. GKLF was found to be expressed extensively in the gastrointestinal tract, and overexpression of GKLF in transfected fibroblasts results in an inhibition of DNA synthesis (7, 21). These data indicate that GKLF may play an important role in the regulation of cell growth. The current study was undertaken to examine the expression of GKLF in normal colon, adenomatous polyps, and tumors. The role of GKLF in the growth and differentiation of colonic epithelium was investigated as well.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Subject population. The expression of GKLF in normal colon, colonic polyps, or tumors was examined in this study. Study participants were drawn from patients who underwent colonoscopy examination at the Boston Veterans Affairs Medical Center (BVAMC). Participants entered the study if they agreed to have biopsy specimens taken from their large bowel during the course of a clinically indicated colonoscopy. Two biopsy specimens were taken from either polyps or tumors and adjacent normal-appearing colonic mucosa and were used for RNA extraction. The study was approved by the BVAMC Research and Development Committee. The tissue specimen from each polyp or tumor was reviewed by a pathologist and classified as malignant, adenomatous, or nondysplastic based on standard criteria.

GKLF antibodies. A peptide was synthesized based on the human GKLF sequence (amino acids 30-47) and was used to raise antiserum (27). This peptide displayed no sequence homology to other known proteins on computer-based analysis. The peptide was conjugated to KLH and injected into New Zealand White rabbits subcutaneously (Covance Research Product, Denver, PA). To characterize GKLF antisera, GKLF and EKLF proteins were prepared from in vitro transcription and translation of GKLF and EKLF cDNAs (EKLF cDNA was kindly provided by Dr. Bieker, Mount Sinai School of Medicine) in the presence of [35S]methionine (0.2 mCi/ml; Amersham) using a rabbit reticulocyte lysate system (TNTT7 quick coupled transcription/translation system, Promega).

Immunoprecipitation of GKLF antiserum and [35S]methionine-GKLF or EKLF protein was performed in PBS (pH 7.4) containing 0.1% Triton X-100. This was prepared by incubating 5 µl (300,000 dpm) of [35S]methionine protein in the PBS/0.1% Triton X-100 buffer with a 1:500 dilution of GKLF antiserum. The mixture was incubated at room temperature for 30 min with continuous mixing and then added to 50 µl of a 50% slurry of protein A-Sepharose 6 MB beads (Pharmacia) and the incubation continued for 2 h. The beads were washed twice in PBS/0.1% Triton X-100 before the addition of 50 µl of sample buffer (2% SDS containing 10% beta -mercaptoethanol) in preparation for SDS-10% PAGE. The gels were dried, and the location of [35S]methionine protein was determined after exposure to X-ray film for 24 h.

To further characterize GKLF antiserum and to exclude potential cross-reactivity between GKLF antiserum and other tissue proteins, the stomach, small intestine, and colon tissues were obtained from Sprague-Dawley rats and subjected to Northern and Western blot analyses (as described below) for the presence of GKLF mRNA and protein in those tissues.

Immunohistochemistry. Tissue sections from normal colon, adenomatous polyp, and colon cancer were deparaffinized, rehydrated, and immersed in 3% hydrogen peroxide-methanol solution for 10 min at room temperature to inhibit endogenous peroxidase activity. The anti-GKLF antibody was diluted in 0.2% crystalline grade BSA in PBS, and slides were incubated in a humidified chamber at 4°C for 16-18 h. The sections were sequentially incubated with biotinylated goat anti-rabbit IgG, streptavidin-horseradish peroxidase (Pharmingen, San Diego, CA), the chromogenic substrate diaminobenzidine (DAB), and hematoxylin counterstaining with intervening acetate buffer washes. Control slides were prepared in an identical manner, except that the tissue sections were incubated with GKLF antiserum and either 10-4 M GKLF or alkaline phosphatase (Sigma Chemical) peptide.

Northern blot hybridization analysis. Total RNA from tissue specimens or cells was extracted using the acid-phenol method of Chomczynski and Sacchi (4). The RNA was then electrophoresed on a 1.5% agarose-6% formaldehyde gel, and Northern blot hybridization analysis was done using stringent conditions as described previously (24). Hybridization was performed using human GKLF, cyclin A, D1, or E, or cyclophilin (Ambion, Austin, TX) cDNAs radiolabeled with [32P]dCTP, using the Klenow fragment of DNA polymerase I and random oligonucleotides as primers (Promega, WI). The blots were washed under stringent conditions, and autoradiograms were developed after exposure to X-ray film at -70°C, using a Cronex intensifying screen (DuPont).

Cell culture and creation of stable cell lines. To elucidate antiproliferative properties of GKLF, HT-29 cells were stably transfected with either sense or antisense human GKLF cDNAs. HT-29 cells were cultured in the McCoy's 5A medium at 37°C in a 95% air-5% CO2 atmosphere. Medium was supplemented with 10% fetal bovine serum, 100 µg/ml streptomycin, and 100 U/ml penicillin. For transfection, sense (cDNA; +42 to +1899 nucleotides) and antisense (cDNA; +1899 to +42 nucleotides) GKLF cDNAs were subcloned into a pCDNA3 expression vector (Invitrogen, San Diego, CA). This antisense construct has previously been demonstrated to inhibit native GKLF expression (27). HT-29 cells were transfected with sense or antisense GKLF cDNA using the lipofectamine method according to the manufacturer's protocol (GIBCO, Gaithersburg, MD). Cells were then cultured in the presence of G418 (GIBCO) to select neomycin-resistant clones. After 2-3 wk, 20 single, independent clones were randomly isolated from each transfection and tested for GKLF protein level. Clones expressing sense GKLF were selected only if they expressed higher GKLF protein levels than wild-type HT-29 cells. Conversely, clones expressing antisense GKLF were selected for their reduced GKLF protein concentrations.

To examine the effect of short-chain fatty acid on GKLF expression, HT-29 cells were fed with 5 mM sodium butyrate (Sigma Chemical) for 1, 3, 5, and 7 days. For control, uninduced cells were cultured in the presence of fresh media for the same period of time. Cell extracts were subjected to Western and Northern blot analyses for the presence of proliferating cell nuclear antigen (PCNA) protein, GKLF, and cyclophilin mRNAs. The specific activity of alkaline phosphatase in the HT-29 cells was determined as an index of differentiation.

[3H]thymidine incorporation. For [3H]thymidine incorporation, wild-type HT-29 cell and HT-29 cell stably overexpressing sense or antisense GKLF DNA were incubated with 1 µCi/ml [3H]thymidine (20 Ci/mmol) at 37°C for 4 h before harvesting. After washing twice with cold PBS, cells were fixed with 10% TCA at 4°C for 30 min, rinsed with 10% TCA, solubilized with 1 N NaOH, and neutralized with HCl. Aliquots equal to 0.1 volume of the solubilized material were counted in triplicate by liquid scintillation. Dishes that contained no cells were labeled and counted to provide background counts.

DNA content analysis. To explore a potential role of GKLF in the cell cycle progression, wild-type and sense GKLF-transfected HT-29 cells were collected and fixed in 70% ethanol for 30 min. The cells were then centrifuged at 800 g for 5 min, and the ethanol was thoroughly removed. After addition of 40 µg/ml of RNase A (Sigma Chemical) and 20 µg/ml of propidium iodide (Sigma Chemical), cell suspensions were incubated in the dark for 30 min at room temperature. The fluorescence of an individual cell was measured in a fluorescence-activated flow cytometer (FACScan, Becton Dickinson, San Jose, CA), using the appropriate dichroic mirror and emission filter. The data were analyzed using Acqcyte software (Phoenix Flow System, San Diego, CA). The Multicycle program (Phoenix) was used for analysis of cell cycle distribution.

Luciferase and beta -galactosidase measurements. To examine transcriptional regulation of cyclin D1 (CD1) and cyclin A (CA) promoters by GKLF, the CD1 promoter (1745 bp upstream from the transcription initiation site) and the CA promoter (3200 bp upstream from the transcription initiation site) were ligated to the pA3-Luc plasmid containing a firefly luciferase reporter gene (26). HT-29 cells were transiently transfected with pCMV galactosidase and CD1-Luc, CA-Luc, or pA3-Luc DNAs in the presence of GKLF or control pCDNA3 plasmid. For luciferase assays, cells were washed twice with PBS and then lysed in 500 µl of lysis buffer following the manufacturer's instructions (Analytical Luminescence, San Diego, CA). To assay luciferase activity, we mixed 100 µl of the cell lysate with 100 µl of luciferase substrate solution A (Analytical Luminescence). Using a luminometer with automatic injection, we then added 100 µl of substrate solution B (Analytical Luminescence) and measured luciferase activity as the light emission over a 30-s period.

beta -Galactosidase activity in 40 µl of the cell lysate was determined after a 5- to 30-min incubation at 37°C with 2 mM chlorophenol red beta -galactopyranoside (Boehringer Mannheim) in 2 nM MgCl2, 0.1 mM MnCl2, 45 mM 2-mercaptoethanol, and 100 mM NaHPO4, pH 8.0. The reactions were stopped by adding 500 µl of 0.5 M EDTA, pH 8.0, and the absorbance at 570 nm was measured using a spectrophotometer. With each experiment, luciferase activity was determined in duplicate and normalized to beta -galactosidase activity for each dish.

Western blot analysis. SDS-PAGE was performed according to the method of Laemmli (14). Protein samples were dissolved in loading buffer (60 mM Tris · HCl, pH 6.8, 2% SDS, 100 mM dithiothreitol, and 0.01% bromphenol blue), heated to 100°C for 3 min, and loaded onto the gel in electrophoresis buffer containing 25 mM Tris · HCl, pH 8.3, 250 mM glycine, and 0.1% SDS. At the completion of electrophoresis, proteins were transferred to a nitrocellulose membrane (Hybond enhanced chemiluminescence, Amersham Life Science). The membrane was incubated overnight in the blocking buffer (10 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Tween 20) containing 5% nonfat powdered milk. The membrane was immunoblotted with CD1 or PCNA antiserum (Santa Cruz Biotech, Santa Cruz, CA). After incubation with the secondary antibody, the membrane was visualized with enhanced chemiluminescence (Amersham).

CD1-associated kinase activity. CD1-associated kinase activities were examined in wild-type and stable HT-29 cells. Cells were lysed in ice-cold modified radioimmunoprecipitation assay (RIPA) buffer (0.1% SDS, 1% Nonidet P-40, 1% sodium deoxycholate, 0.15 M NaCl, 10 mM sodium phosphate, pH 7.0, 100 µM sodium vanadate, 50 mM sodium fluoride, 50 µM leupeptin, 1% aprotinin, 2 mM EDTA, and 1 mM dithiothreitol). The samples were then sonicated and centrifuged in the cold room for 10 min, and the supernatants were collected and stored at -80°C. Total protein (100-500 µg) was subjected to immunoprecipitation with the CD1 antibodies. The immunocomplexes were collected with protein A-agarose and washed four times with RIPA buffer and three times with kinase buffer (50 mM Tris, pH 7.4, 10 mM MgCl2, and 1 mM dithiothreitol). Twenty-five microliters of kinase buffer with purified glutathione-S-transferase-retinoblastoma (GST-Rb) protein, 10 mM ATP, and 10 µCi [gamma -32P]ATP (3,000 Ci/mmol, Amersham Pharmacia Biotech) were added, and the reactions were incubated at room temperature for 30 min. The reaction was stopped by addition of 20 µl of 5× Laemmli buffer, boiled for 10 min, and separated on a 12.5% acrylamide gel. The amount of radioactivity incorporated was analyzed by autoradiography and densitometry.

Alkaline phosphatase assay. After culture, cells were lysed with 0.25% sodium deoxycholate and total protein was determined (3, 12). Standard alkaline phosphatase assay (ALP) solutions ranging from 0 to 70 U/ml were prepared (Sigma enzyme control). One-hundred-microgram aliquots of each sample and standards were measured by adding 7 mM p-nitrophenyl phosphate, 0.1 mM NaHCO3, and 5 mM MgCl2 and then incubated at 37°C for 25 min with protection from light. Reactions were terminated by addition of 1 ml of 0.1 N NaOH to each tube. Enzyme concentrations were determined by colorimetric assay measuring the absorbance at 410 nm and calculated from the standard curve. Each sample was assayed in duplicate.

Statistics. Results were expressed as means ± SD. Statistical analysis was performed using ANOVA and Student's t-test. P < 0.05 was considered to be statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
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RESULTS
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Expression of GKLF transcript in normal colon, adenomatous polyps, and cancer. Northern blot analysis was performed on total RNA isolated from colonic tissue using a 32P-labeled probe prepared from human GKLF or cyclophilin cDNA. A representative Northern blot autoradiogram is shown in Fig. 1, where a major 3.5-kb band was noted for GKLF. This band is consistent with the predicted size published previously (7, 21, 27). The levels of GKLF mRNA, expressed as the ratio of GKLF mRNA to cyclophilin mRNA to correct for gel loading, were significantly lower in cancer and adenomatous polyps than in normal colon (42 ± 18% and 64 ± 20% of normal control, respectively; P < 0.05; Fig. 2).


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Fig. 1.   Representative Northern blot autoradiograms used to measure gut-enriched Krüppel-like factor (GKLF) (top) and cyclophilin (bottom) mRNA levels. Five micrograms of total RNA extracted from biopsy specimens was subjected to Northern blot analysis using human GKLF and cyclophilin cDNA probes. N, normal colonic tissue; T, adenocarcinoma; A, adenomatous polyps.



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Fig. 2.   Steady-state levels of GKLF mRNA in normal colon, carcinoma, and adenomatous polyps. GKLF mRNA concentrations in the polyps or tumors were expressed as % of normal control tissue and calculated as means ± SD of the ratio of GKLF mRNA to cyclophilin mRNA to correct for gel loading; n = 6-8 for each group. * P < 0.05 compared with normal colon.

Characterization of GKLF antiserum. To examine the specificity of GKLF antiserum, GKLF and EKLF proteins prepared from in vitro transcription and translation were immunoprecipitated with anti-GKLF antibodies. As demonstrated in Fig. 3, a major 60-kDa band was visualized in GKLF but not in EKLF cDNA transcribed protein, indicating the lack of cross-reactivity between GKLF antiserum and EKLF protein. In rat tissues, GKLF mRNA message was detected in the gastric fundus, duodenum, jejunum, ileum, and colon (Fig. 4A). These findings were confirmed by the presence of GKLF protein in Western blot analysis (Fig. 4B). Together, these data support the specificity of GKLF antiserum.


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Fig. 3.   SDS-PAGE analysis of GKLF (lane A) and erythroid Krüppel-like factor (EKLF) (lane B) proteins immunoprecipitated with anti-GKLF antiserum. GKLF and EKLF proteins were synthesized from in vitro transcription and translation of each individual cDNA as described in MATERIALS AND METHODS. Size standards are indicated at left.



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Fig. 4.   Expression of GKLF in the rat gastrointestinal tract. Twenty micrograms of total RNA and 40 µg of protein from fundus (lane 1), duodenum (lane 2), jejunum (lane 3), ileum (lane 4), and colon (lane 5) were subjected to either Northern (A) or Western (B) blot analysis using 32P-labeled GKLF cDNA or GKLF antiserum as probes. Size standards for RNA and protein are indicated at left.

Immunolocalization of GKLF in colon. Histology sections from normal colon, adenomatous polyp, and colon cancer were examined for the expression of GKLF using antiserum characterized above. In normal colon (Fig. 5, A, C, and G), GKLF protein was expressed primarily in the epithelium, and its level was higher at the surface epithelium and gradually decreased toward the crypt. Few GKLF-stained cells were scattered in the lamina propria. GKLF immunostains were predominantly cytoplasmic at the normal surface epithelium (Fig. 5, A and C) but were present in the cytoplasm and the nucleus of crypt epithelium (Fig. 5, A, C, and G). Moreover, GKLF immunostains in the crypt epithelium appear to be heterogeneous with some cells staining negative for GKLF. In adenomatous polyp, the location of GKLF immunostain was mainly nucleic and no surface-to-crypt gradient was observed (Fig. 5H). GKLF immunostains in colon cancer were diffusely distributed in the majority of cancer cells (Fig. 5, D and F). GKLF immunostains in normal and cancerous tissues were abolished by blocking sections with GKLF (Fig. 5, B and E) but not with the nonspecific peptide alkaline phosphatase (Fig. 5, C and F), demonstrating the specificity of the GKLF immunostains. Similar results were observed in the adenomatous mucosa (data not shown).


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Fig. 5.   Immunolocalization of GKLF in the colon. Adjacent sections of normal colon (A, B, C, and G), adenomatous polyp (H), and adenocarcinoma (D-F) were stained with GKLF antiserum (1:100). GKLF immunostain (brown) in the normal colon was localized primarily on the surface epithelium (A and C; arrowhead), and its level gradually decreased toward the crypt cells (A, C, and G; single arrow). GKLF immunostain was diffusely distributed in the cancerous tissue (D and F; double arrow) and visualized mainly in the nucleus of the adenomatous epithelia (H, single arrow). GKLF immunostain was completely abolished when tissue sections were blocked with GKLF peptide (B and E) but not affected by the nonspecific peptide alkaline phosphatase (C and F). Original magnification: ×100 for A-F and ×150 for G and H.

Effect of GKLF expression on DNA synthesis. The results of the above studies suggest that downregulation of GKLF in the colonic epithelium may result in uninhibited cell growth and malignant transformation. To assess the effect of GKLF on cell growth, wild-type HT-29 cells and HT-29 cells stably expressing sense or antisense GKLF cDNA were cultured in the absence of serum for 24 h. Cells were then exposed to media containing 10% fetal bovine serum for 4 h, at which time DNA synthesis was determined by [3H]thymidine incorporation. Figure 6 shows the results of five independent experiments. Overexpression of sense GKLF in HT-29 cells resulted in a 66 ± 12% decrease in the [3H]thymidine uptake, whereas downregulation of GKLF expression in antisense-transfected cells led to a 25 ± 11% increase in [3H]thymidine incorporation compared with wild-type cells.


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Fig. 6.   %Change of [3H]thymidine incorporation in wild-type HT-29 and HT-29 cells stably transfected with sense or antisense GKLF cDNA. Data were expressed as %change over wild-type cells, and data represent means ± SD of 5 separate experiments with each value determined in triplicate. * P < 0.05, ** P < 0.01 compared with wild-type control.

Effect of GKLF on cyclin expression. The data from preceding studies suggest that the expression of GKLF is temporally associated with cell growth. To examine whether these growth effects were associated with changes in cyclin expression, the levels of CD1, cyclin E, and cyclin A mRNA were examined in wild-type and sense- or antisense-transfected HT-29 cells. As shown in Fig. 7, CD1 mRNA levels were significantly decreased in sense GKLF expressed cells, but increased in antisense GKLF-transfected cells, with levels of 48 ± 25% and 135 ± 17%, respectively of the wild-type control. No significant change in the expression of cyclin A, cyclin E, or cyclophilin transcript was observed in those cells (Fig. 7).


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Fig. 7.   Representative Northern blot autoradiograms from 5 independent experiments used to measure cyclin D1 (CD1), cyclins A and E, and cyclophilin mRNA levels in wild-type HT-29 cells (lane A) and HT-29 cells stable transfected with sense (lane B) or antisense (lane C) GKLF cDNA. Twenty micrograms of total RNA from each cell line were subjected to Northern blot analysis using specific probes.

CD1 protein and CD1-associated kinase activity. To examine the molecular mechanisms responsible for GKLF-mediated growth arrest, CD1 protein and CD1-associated kinase activity were measured in wild-type and GKLF-transfected HT-29 cells. As shown by Western blot analysis, CD1 protein levels were significantly decreased in sense GKLF-expressed cells but increased in antisense GKLF-transfected cells (Fig. 8). Similar changes in CD1-associated kinase activity, as determined by GST-Rb phosphorylation, were observed in those cells. These results indicate that the growth arrest effect of GKLF is associated with the decrease in CD1 protein level and in CD1-associated kinase activity.


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Fig. 8.   Autoradiograms of representative CD1 protein (top) and CD1-cyclin-dependent kinase phosphorylation of GST-Rb in wild-type HT-29 cells (lane A) and HT-29 cells stably transfected with sense (lane B) or antisense (lane C) GKLF cDNA. Forty micrograms of protein from each cell line were resolved on SDS-PAGE gels, transferred to a membrane, and blotted with anti-CD1 antibodies to measure CD1 protein levels. To determine CD1-associated kinase activity, CD1 immunoprecipitates from cell extracts were assayed for kinase activity in the presence of GST-Rb and [gamma -32P]ATP (bottom) as described in MATERIALS AND METHODS. These data are representative of 4 independent experiments.

Effect of GKLF on cell cycle progression. In mammalian cells, CD1 is normally expressed during the G1 interval and is believed to be an important factor in driving cells through a G1 restriction and entering S phase. To explore the effect of GKLF on cell-cycle evolution, wild-type and sense GKLF-transfected HT-29 cells were subjected to cell-cycle analysis. As shown in Fig. 9, compared with wild-type cells, overexpression of GKLF resulted in an increase in cells arrested at G1 phase, suggesting a potential role for GKLF in modulating cell cycle progression.


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Fig. 9.   Cell cycle phases of wild-type HT-29 cells (A) and HT-29 cell overexpressing sense GKLF cDNA (B). DNA content from each cell line was analyzed by fluorescence-activated flow cytometry. %Cells in G1 phase is shown at right. Data represent means ± SD of 5 separate experiments. Abscissa, DNA content; ordinate, cell number.

Inhibition of CD1 promoter activity by GKLF. To determine the mechanism(s) by which GKLF inhibited CD1 mRNA expression, the effect of GKLF on CD1 promoter activity was further assessed. HT-29 cells were transiently transfected with CD1-Luc or CA-Luc (2 µg/well) reporter plasmid and pCMV beta -galactosidase DNA (0.1 µg/well; control for transfection efficiency) in the presence or absence of GKLF DNA (0.05 µg/well). As shown in Fig. 10, transfection of HT-29 cells with CD1-Luc DNA increased luciferase activity to ~100-fold over the promoterless control (pA3-Luc), and this activity was significantly repressed by GKLF (90%). In contrast, GKLF had no effect on either the CA promoter or the basal reporter activity, indicating the specificity of its inhibitory effect on the CD1 promoter.


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Fig. 10.   Effect of GKLF on transcriptional activity of CD1 (CD1-Luc), cyclin A (CA-Luc), and promoterless reporter (pA3-Luc) plasmids in HT-29 cells. Promoter constructs, pCMV galactosidase, and GKLF or control pCDNA-3 plasmids were transfected into HT-29 cells. After incubation for 48 h, the cells were harvested for analyses of luciferase and beta -galactosidase activities. Data represent means ± SD of 3 separate transfections after correcting for differences in transfection efficiencies by beta -galactosidase activities. * P < 0.01 compared with control (pCDNA3).

Increased GKLF mRNA levels in differentiated HT-29 cells. To explore the potential role of GKLF in modulating differentiation of the colonic mucosa, the expression of GKLF mRNA message was examined in HT-29 cells during the short-chain fatty acid-promoted differentiation process (18). In the presence of sodium butyrate, HT-29 cells exhibited more differentiated phenotypes as indicated by the increase in alkaline phosphatase activities and the decrease in PCNA levels from day 3 to day 7 (Fig. 11). GKLF mRNA levels were gradually increased as cells became more differentiated (Fig. 11). Moreover, the basal alkaline phosphatase levels in HT-29 cell overexpressing sense GKLF appeared to be higher then those in wild-type HT-29 cells (28 ± 6 and 12 ± 5 U/100 µg protein, respectively). These data indicate that GKLF may also play an essential role in regulating differentiation of the colonic epithelium.


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Fig. 11.   Potentiation of differentiation in HT-29 cells by short-chain fatty acid. HT-29 cells were cultured in the presence of 5 mM sodium butyrate for 1, 3, 5, and 7 days. Cells from the culture plates were collected to determine alkaline phosphatase (ALP) activity, proliferating cell nuclear antigen (PCNA) protein levels and GKLF and cyclophilin mRNA concentrations. Twenty micrograms of total RNA and 40 µg of protein from cell lysates were subjected to Northern and Western blot analyses. Specific activity of ALP in each day was expressed as means ± SD of at least 3 independent experiments. * P < 0.05 compared with day 0.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although many genetic alterations have been attributed to the development of colon cancer, the early events leading to colonic carcinogenesis are not clear. Moreover, hyperproliferation of the colonic mucosa has been observed to precede the formation of colonic polyps or tumors, but the mechanisms mediating cellular hyperproliferation have not been fully elucidated. In this report, we have demonstrated that the expression of GKLF mRNA is significantly decreased in neoplastic colonic tissues including adenoma and carcinoma. These results are consistent with findings from Ton-That et al. (23), who demonstrated that the level of GKLF transcript is decreased in the intestine of multiple intestinal neoplasia mice during a period of tumor formation. Together, these data suggest that downregulation of GKLF expression may contribute to malignant transformation of the colon.

Previous studies (4, 5) using in situ hybridization have shown that GKLF is expressed extensively in the gastrointestinal tract. By Northern blot analysis, we also found that GKLF mRNA was present in the rat stomach, small intestine, and colon. Despite these findings, whether GKLF exhibits any significant role in modulating cell growth in the gastrointestinal tract has not been reported. In current study, constitutive overexpression of GKLF in human adenocarcinoma cells resulted in a decrease in [3H]thymidine uptake, whereas inhibition of GKLF expression led to an increase in DNA synthesis, suggesting that GKLF may play an essential role in controlling growth arrest in the colon. Furthermore, these results also imply that downregulation of GKLF may result in uninhibited cell growth and are consistent with findings from Shields et al. (21), who showed that GKLF mRNA levels in NIH 3T3 cells were significantly decreased when cells were rendered to proliferate (21).

The mechanism of GKLF-mediated growth arrest in the colonic epithelium is currently unknown. The eukaryotic cell cycle, consisting of four major phases (G1, S, G2, and M), is a series of carefully regulated events (10). When quiescent cells are stimulated, a cascade of cellular events takes place, resulting in DNA synthesis and subsequent cell division (10, 15, 16, 22). In addition, eukaryotic cells also possess proteins, such as tumor suppressor p53, which exhibit negative control on cell growth (9). Recently, another group of genes (p21, p27, p16, and p15) was found to inhibit the activities of the cyclin and cyclin-dependent kinases, both of which are essential for the progression of the cell cycle (20). In this study, we have shown that the expression of GKLF is closely related to that of CD1 and CD1-associated kinase activity. Moreover, GKLF suppresses the CD1 promoter activity. Although the causative role of CD1 in the growth inhibitory effect of GKLF has not yet been established, our results suggest that the function of GKLF may be mediated, at least in part, through the repression of the CD1 gene. This notion is further supported by the identification of multiple potential GKLF binding domain, CACCC motif, on the CD1 promoter (26). Whether GKLF regulates the expression of other cyclins or cyclin-dependent kinases warrants further investigation. Moreover, in contrast to our findings, Jenkins et al. (13) recently reported that GKLF increased transcriptional activity of the human keratin 4 and Epstein-Barr virus ED-L2 promoters and suggested that GKLF might function as a transcriptional activator in the esophageal squamous epithelium to regulate cell differentiation. Yet et al. (27) recently provided evidence showing that GKLF possessed both transcriptional activation and repression domains. It is feasible that GKLF may function as either transcriptional activator or repressor and that this property is promoter or cell specific.

Cancer cells differ from normal cells in many characteristics, including loss of differentiation, which arises from uninhibited cell growth and uncontrolled cellular evolution (9). In the dysplastic colonic epithelium, GKLF mRNA levels were significantly decreased. In contrast, GKLF mRNA levels increase as HT-29 cells gain more differentiated phenotype in response to short-chain fatty acid stimulation. These findings suggest that GKLF may also play a role in controlling the switch from proliferation to differentiation in the colonic epithelium. It is possible that upregulation of GKLF is essential for colonic cells to become differentiated and that downregulation of GKLF allows cells to enter the cell cycle and result in uncontrolled cell growth. These hypotheses warrant further exploration.

In the normal colonic mucosa, GKLF immunostain was found to locate primarily in the mature surface epithelium, and its level of expression gradually decreased toward the crypt cells. When colonic epithelia become dysplastic, as seen in the adenomatous or cancerous tissues, the surface-to-crypt gradient of GKLF immunostain disappears. These findings are consistent with a recent report from Foster et al. (6), who demonstrated that, in oral squamous mucosa, the expression of GKLF was predominantly located in the differentiating epithelia and that GKLF immunostains were diffusely distributed when cells became dysplastic. Foster et al. (6) suggested that misexpressed GKLF in the basal compartment of the squamous epithelium may result in tumorigenesis. This conclusion is similar to our findings showing that GKLF immunostains appear to localize in the proliferating epithelium of colonic adenomatous polyp and cancer. Whether aberrant expression of GKLF results in hyperproliferation of colonic epithelium requires further investigation. Moreover, the immunostain of GKLF in the crypt appears to be heterogenous. Whether these cells stained positive for GKLF represent active proliferating cells also require further study.

In summary, the results of this study suggest that GKLF may play an important role in modulating the switch of proliferation and differentiation in the colonic epithelia and that its level of expression is reduced in the adenomatous polyps and cancers. Furthermore, downregulation of GKLF results in uninhibited cellular growth that may contribute to malignant transformation of the colonic mucosa. Finally, GKLF appears to be a transcriptional repressor of the CD1 gene.


    ACKNOWLEDGEMENTS

This work was supported by United States Public Health Service Grants DK-52186 (C.-C. Tseng) and R29-CA-70897 and RO1-CA-75503 (R. G. Pestell). R. G. Pestell is a recipient of the Irma T. Hirschi Award and an award from the Susan G. Komen Breast Cancer Foundation. Work conducted at the Albert Einstein College of Medicine was supported by Cancer Center Core National Institute of Health Grant 5-p30-CA-13330-26.


    FOOTNOTES

Address for reprint requests and other correspondence: C.-C. Tseng, Section of Gastroenterology, Boston Univ. School of Medicine, Boston, MA 02118. (E-mail: chichuan.tseng{at}bmc.org).

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

Received 2 September 1999; accepted in final form 2 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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