Induction of DMBT1 expression by reduced ERK activity during a gastric mucosa differentiation-like process and its association with human gastric cancer

Weiqun Kang 1, 4, Ole Nielsen 2, Claus Fenger 2, Graham Leslie 3, Uffe Holmskov 3 and Kenneth B.M. Reid 1, *

1 MRC Immunochemistry Unit, Department of Biochemistry, Oxford University, Oxford OX1 3QU, UK and 2 Department of Pathology and 3 Department of Immunology and Microbiology, Institute of Medical Biology, University of Southern Denmark, DK-5000 Odense C, Denmark
4 Present address: Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA

* To whom correspondence should be addressed. Tel: +44 1865 275 353; Fax: +44 1865 275 353; Email: kenneth.reid{at}bioch.ox.ac.uk


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Abnormalities in the expression of DMBT1 (deleted in malignant brain tumors 1) have been implicated in the development of esophageal, gastric and colorectal cancers of the alimentary tract, but the underlying mechanism remains unclear. In the present study, using the gastric cell line AGS, we identified two intracellular signaling molecules protein kinase C (PKC) and extracellular signal-related kinase (ERK). They mediated both the phorbol myristate acetate (PMA) downregulation of DMBT1 expression and the initiation of cell differentiation, which was measured by cell cycle withdrawal and the induction of the tissue-specific marker trefoil factor 1 (TFF1). A time-course study showed that following the PMA activation of ERK kinase, the induction of TFF1 and the reduction of DMBT1 were detected at the same time point. We then demonstrated a minimal level of DMBT1 in proliferating AGS cells seeded at low density, where ERK activity was high. Reduction of ERK activity, either by an ERK inhibitor PD98059 or by high-density seeding, significantly reduced AGS cell growth judged by CFSE labeling. This cellular effect was elicited by cyclin D/p21 (Cip/Waf1) and G0/G1 arrest, and was accompanied by a marked increase in DMBT1-expressing cells. Finally, we showed that siRNA directed against DMBT1 had no effect on the induction of a cell growth arrest marker, gut-enriched Kruppel-like factor (GKLF), but reduced the PMA induction of TFF1. Along with its upregulation coinciding with G0/G1 arrest, and its attenuation in differentiated cells, these results suggest that the transient induction of DMBT1 is apparently specific at an early stage of gastric epithelial differentiation-like process, when it may play a role in cell fate decision. Consistent with such a potential function, we detected frequent abnormalities of the DMBT1 expression in the specimens of human gastric adenocarcinoma.

Abbreviations: CFSE, 5-,6-carboxyfluorescein diacetate succinimidyl ester; DMBT1, deleted in malignant brain tumors 1; DMSO, dimethyl sulfoxide; ERK, extracellular signal-related kinase; GKLF, gut-enriched Kruppel-like factor; MEK, MAP kinase kinase; mAbs, monoclonal antibodies; PBS, phosphate-buffered saline; PMA, phorbol myristate acetate; PKC, protein kinase C; PI, propidium iodide; RT–PCR, reverse transcriptase–polymerase chain reaction; TFF1, trefoil factor 1; TfR, transferring receptor


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
DMBT1 (deleted in malignant brain tumors 1) is a gene that is located at chromosome 10q 25.3–26.1, a possible tumor suppressor locus indicated by the refinement of the losses of heterozygosity in various cancers (1). The repetitive structure of the gene has been proposed to increase its susceptibility to genomic instability, which leads to the homozygous/hemizygous deletion observed in a significant number of medulloblastomas and glioblastoma multiforme (1,2). Abnormalities of its expression in brain, lung and gastrointestinal tumors have also been reported (37). Based on these findings, DMBT1 was considered as a candidate tumor suppressor gene (1,2,7). However, studies of the functions of this molecule and its relationship with tumorigenesis are still at an initial stage.

Rabbit hensin and mouse CRP-ductin represent the rodent orthologs of DMBT1 (3,8). The two molecules are implicated in epithelial differentiation, in the kidney duct and small intestine, respectively (9,10). Al-Awqati et al. (9) further showed that the deposition of hensin into the extracellular matrix and its polymerization by galectin 3 were necessary for such a function. Co-localization of DMBT1 with hensin and CRP-ductin in the gastrointestinal tract and the kidney (3,8), and its characteristic spatial and temporal distribution in fetal and adult epithelia (3), suggest that DMBT1 may share a similar function with its counterparts in other species.

To address this possibility, we previously used the gastric cell line AGS to demonstrate a marked downregulation of DMBT1 upon phorbol myristate acetate (PMA) treatment, which was seen in parallel to the induction of differentiation-specific expressions in these cells (11). In the present study, our aims were 3-fold: first, to identify intracellular signaling molecules which may mediate the PMA regulation of DMBT1 expression; second, using different culture conditions to analyze extracellular signal-related kinase (ERK) activation-related changes, in DMBT1 level and events related to cell proliferation and differentiation; third, to analyze the DMBT1 distribution in human gastric adenocarcinoma samples. Our results suggested an association between DMBT1 expression and gastric epithelial differentiation-like process, and the potential for this molecule to serve as a biomarker in gastric cancer.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Reagents
PMA (Calbiochem, La Jolla, CA) was dissolved in dimethyl sulfoxide (DMSO) to make a 2 mg/ml stock solution. PD98059 (Calbiochem), a selective and cell-permeable inhibitor of MAP kinase kinase (MEK), was dissolved in DMSO at 10 mg/ml. A stock solution of 1-(5-isoquinolonesulfonyl)-2-methylpiperazine (H7, Calbiochem), one of the H series inhibitors against protein kinase C (PKC), was prepared in water at 25 mg/ml. A 50 µM stock solution of the stable intra-cytoplasmic dye, 5-,6-carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes, Leiden, The Netherlands) was prepared in DMSO. A ribonuclease A (RNase, Sigma, St Louis, MO) solution was prepared by dissolving it (1 mg/ml) in phosphate-buffered saline (PBS). All the above solutions were stored in appropriate aliquots at –20°C. Propidium iodide (PI, Sigma) was supplied as a 400 µg/ml solution in water and stored in darkness at 4°C.

Monoclonal antibodies (mAbs) against DMBT1/gp-340 (Hyb 213-6) has been described previously (12,13). Monoclonal antibodies against cyclin D3, p21 (Cip/Waf1) and Ki67 were obtained from Calbiochem. The polyclonal antibody against trefoil factor 1 (TFF1) (2239A) was a kind gift from Lars Thim (Novo Nordisk, Denmark). EnVision+System (DAB) was purchased from Dako (Glostrup, Denmark).

Cell culture and treatments
AGS (American Type Culture Collection, Rockville, MD), a poorly differentiated gastric adenocarcinoma cell line, was grown in F-12K nutrient mix (Life Technology, San Diego, CA) containing 10% (v/v) fetal bovine serum, 100 µg/ml streptomycin and 100 IU/ml penicillin. All cultures were maintained at 37°C in a humidified atmosphere containing 95% air and 5% CO2.

For stimulation, a confluent monolayer of AGS cells were cultured in 3.5 ml medium in 6-well polystyrene tissue culture plates (Corning Costar, Cambridge, MA) or 8-chamber slides (Nunc, Naperville, IL) for immunocytochemical staining. PD98059 was diluted in culture medium and added in a concentration gradient to a maximum of 50 µM; H7 was added at 15, 30 and 60 µM. Both reagents were applied to culture 1 h before PMA treatment (50 ng/ml) for various time periods. Cells were washed three times with PBS and harvested with trypsin [0.05% (w/v), Life Technology] and EDTA (0.02%, w/v) for proliferation assay or cell cycle analysis, or lysed directly in each well for total RNA isolation.

siRNA experiment
AGS cells were plated in 6-well plates at a density of 4 x 105 cells/well and allowed to attach overnight. The cells were then transfected with an siRNA duplex directed against nucleotides 6604–6624 of DMBT1 mRNA (accession No. NM_007329) (5' AAU AUC ACC AGG UUC GGC G 3') or a mismatch siRNA duplex (5' AAU AUC AUU AGG UUC GGC G 3') synthesized by Qiagen (Valencia, CA). Transfection was performed using Oligofectamine reagent (Invitrogen, Carlsbad, CA), following the manufacturer's instruction. Following transfection, the cells were harvested before reaching confluence, or reached confluence after overnight growth. They were then either treated with PMA (50 ng/ml) or left to further grow for 24 h.

Reverse transcriptase–polymerase chain reaction (RT–PCR)
Expressions of DMBT1, TFF1 and gut-enriched Kruppel-like factor (GKLF) mRNAs were determined by RT–PCR as described previously (11). In brief, total RNA samples were isolated from cells under various culture conditions by the use of an RNeasy kit (Qiagen, West Sussex, UK). The respective total RNAs (5 µg) were converted into cDNAs using Oligo (dT)15 primers and Superscript II reverse transcriptase (Life Technology). These cDNA preparations were subjected to PCR. The primer sequences and PCR conditions for DMBT1 and TFF1 were as described previously (11). The primer pair for GKLF is forward: 5'-GGCGCTGGACCCCCTCTC-3', and reverse: 5'-GCAGCCCGCGTAATCACAAGT-3'. All the primers were designed to cross exon–intron borders, to exclude the amplification of genomic templates. Human transferrin receptor (TfR) was used to standardize the amount of template for specific primers. The PCR products of DMBT1, TFF1, GKLF and TfR were subjected to agarose gel electrophoresis. The bands were extracted using a QIAEX II gel extraction kit (Qiagen) and sequenced.

Western blotting
AGS cells with different treatments were scraped into M-per lysis buffer (Pierce, supplemented with protease and phosphatase inhibitors). Thirty micrograms of total cell extracts from each treatment were subjected to SDS–PAGE [4–12% (w/v), acrylamide] followed by an electrotransfer onto PVDF membranes. Phosphorylated ERK was detected by immunoblotting using Phospho-p44/42 MAP Kinase (Thr202/Tyr204) antibody (Cell signaling). The signals were visualized with the ECL detection system (Amersham). The membrane was stripped and re-blotted with p44/42 MAP Kinase antibody (Cell signaling) to determine the input.

Immunocytochemistry
Cells were washed twice with PBS, fixed in 4% (w/v) paraformaldehyde and permeabilized in PBS containing 0.1% (v/v) Triton X-100. Immunostaining was performed with DAKO EnVision+System (DAB) following the manufacturer's instructions. The specific mAbs were added at: anti-DMBT1 (Hyb 213-6), 8.5 µg/ml; anti-cyclin D3 and anti-p21 (Cip/Waf1), 5 µg/ml. Controls were performed by replacing the primary antibodies with an isotype mAb (Sigma) to the highest concentration used for specific antibodies. After brief nuclear counter staining in Mayer's hematoxylin, cover slips were mounted with Aquatex (Merck, Darmstadt, Germany).

Cell proliferation assay
Cell proliferation rate was determined using flow cytometric analysis on CFSE labeled AGS cells. Each daughter cell inherits approximately half of the fluorescent dye from the parental cell, hence allowing the quantification of cell divisions by the progressive decrease of CFSE fluorescence at different time intervals (14). AGS cells were harvested by trypsin digestion, washed three times and resuspended in 2 ml PBS (5 x 106 cells/ml). Forty microliters of CFSE stock solution was applied to 1 ml cells to get a final concentration of 2 µM. Cells were incubated for 10 min during which they were carefully mixed three to four times. The stained cells were washed in serum-free and ice-cold RPMI-1640 medium (Sigma) two times, after which the cells were resuspended in supplemented F-12K medium (density adjusted to the desired level) and further cultured for 24 h, before the addition of PD98059, for another 24 h.

Cell cycle analysis
AGS cells were harvested by trypsinization and washed three times with PBS. The cells were suspended in supplemented F-12K medium containing 4 µg/ml DNase (Type I bovine pancreatic DNase, Sigma) and incubated at 37°C for 10 min, followed by washes in PBS and being resuspended in 200 µl of PBS. An addition of 2 ml of ice-cold 70% ethanol (30% distilled water) was made and left on ice for at least 30 min. The cells were then harvested by centrifugation at 300 g for 5 min and resuspended in 400 µl of PBS, pH 7.3. Fifty microliters each of the RNase and the PI solutions were added and incubated at 37°C for 30 min. After three washes in PBS, the DNA content was measured using Beckman Dickson FACS machine, and the results were analyzed using FlowJo program (Treestar).

Grid counting
The immunocytochemical staining slides were placed on a platform attached to a Leica microscope, which was operated automatically by a CAST-grid system (Olympus, Denmark). The positive/negative stained cells (1000 cells in total for each condition) were counted at x200 magnification, in the fields randomly chosen by the system. The statistical analysis was performed using an origin program.

Immunohistochemistry
The procedure was carried out as described previously (11). Sections of 4 µm were cut from paraffin-embedded blocks of tissue and cell pellets from cultured epithelial cells fixed in neutral-buffered formaldehyde. Sections were mounted on ChemMate Capillary Gap Slides (Dako, Glostrup, Denmark), dried at 60°C, deparaffinized and hydrated. Antigen retrieval was performed using microwave heating in Target Retrieval Solution (Dako). Any endogenous biotin was then blocked with a Biotin-Blocking System (Dako), and the slides were incubated with mAbs against DMBT1 (Hyb213-6, 8.5 µg/ml) or Ki-67 antigen (Dako, 20 µg/ml), and a polyclonal antibody against TFF1 (2239A) in 1:500 dilution, for 25 min at room temperature. Immunostaining was performed with the ChemMate HRP/DAB detection kit (Dako), and was followed by a brief nuclear counterstaining in Mayer's haematoxylin (Dako). The specificity of immunostaining was verified by replacing the primary antibodies with an isotype mAb or irrelevant polyclonal antibody to the same concentration.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Intracellular signal molecules involved in PMA downregulation of DMBT1
In an attempt to search for possible intracellular signaling molecules that mediate the PMA downregulation of DMBT1 in AGS cells, H7, an inhibitor for PKC activation, was identified by RT–PCR to inhibit the downregulatory effect of PMA on DMBT1 transcripts, in a concentration-dependent manner (Figure 1A, a). At the protein level, immunocytochemical staining revealed that DMBT1 disappeared in AGS cells after 24 h of PMA treatment (Figure 1B, b), whereas clear signals were maintained in H7 pretreated cells (Figure 1B, c). Fifty millimicrons of PD98059, a specific inhibitor for MEK activation, also resulted in the recovery of the mRNA production close to the basal level (Figure 1A, b). Quantification by a grid counting procedure demonstrated that ~3.5% of AGS cells were DMBT1-positive 24 h after confluent seeding. PMA reduced this number to 0.21%, which was restored to ~2.7% when PD98059 was applied along with PMA (Figure 1B, e). These results indicated the involvement of the intracellular signal molecules PKC and ERK MAP kinase in the regulation of DMBT1 expression.



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Fig. 1. Inhibition of H7 and PD98059 blocks the PMA downregulation of DMBT1. (A) RT–PCR analysis shows the reversion of the PMA decrease of DMBT1/gp-340 mRNA by H7 (a) or PD98059 (50 µM, b) in AGS cells. A TfR specific product from the cDNAs of all the treatments was used as an internal control. (B) Immunostaining demonstrates the significant decrease of AGS cells stained positive for DMBT1 after PMA treatment (50 ng/ml) (b) compared with control cells (a), and the inhibition by co-incubation with H7 (60 µM, c) or PD98059 (50 µM, d). Original magnification, x200. (e) Bar graph depicts the average percentages of DMBT1 positive cells after 24 h of various treatments (values are means ± SE of three replicate experiments), evaluated by a grid-counting system as described in Materials and methods. (C) Treatment with PMA for 24 h withdraws a significant portion of AGS cells into G0 phase, judged by the increasing percentage of AGS cells stained negative for Ki67 (a and b), which is inhibited by PD98059 co-treatment (c). Original magnification, x200. Each experiment was repeated twice with similar results.

 
PMA leads to the cell cycle withdrawal of AGS cells, which was inhibited by PD98059
Cell cycle withdrawal characterizes terminal differentiation in different types of cells (1517). Less than 5% of AGS cells in confluent-seeding were negative for the nuclear antigen, Ki67 [represented in all stages of the cell cycle except G0 (18,19)], hence withdrawn from the proliferating cycle (Figure 1C, a). PMA treatment for 24 h led to a 6-fold increase (30 ± 5%) of Ki67 negative cells (Figure 1C, b), indicating its ability to drive AGS cells into G0 phase. Addition of PD98059 reduced the PMA-mediated increase to <2-fold (8 ± 2%, Figure 1C, c).

Dynamics of PMA regulation of MEK kinase, DMBT1 expression and the differentiation-specific expression TFF1, all of which were inhibited by PD98059
Time-course studies showed a marked early PMA induction of ERK1/2 phosphorylation (within 30 min), which was blocked by PD98059 (Figure 2A). This pattern lasted for 10 h. Meanwhile, PMA caused a decrease of DMBT1 mRNA, which was first distinguishable at 8 h (Figure 2B), when PD98059 inhibition of the process was also visualized by RT–PCR. Both events reached more substantial levels between 12 and 24 h: a nearly complete PMA suppression of the transcripts was retained in the presence of PD98059. Induction of the TFF1 gene became detectable after 4 h of PMA treatment (no such response was seen at 2 h, data not shown), and became more pronounced after 12 h. At all time points of being analyzed, this induction was significantly inhibited by the co-treatment with PD98059.



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Fig. 2. PMA and PD98059 differential regulation of ERK phosphorylation, gene expression of DMBT1 and differentiation marker TFF1. AGS cells were treated with PMA (50 ng/ml) or PD98059 (50 µM), alone or in combination for indicated time. Cells were extracted for (A) protein to determine ERK phosphorylation, showing PMA induction of ERK activation blocked by PD98059 in confluent-seeding cells; and (B) RNA for RT–PCR analysis of DMBT1 and TFF1 transcripts. TfR specific product was used as an internal control.

 
PD98059 increases transcripts of DMBT1, but not of the differentiation marker TFF1
As one of the controls, the MEK-specific inhibitor PD98059 was added alone to the AGS cell culture, where it blocked the ERK1/2 phosporylation (Figure 2A). Interestingly, its induction of DMBT1 transcripts was observed from 8 h onward. This effect was maximal at 24 h without a significant induction of TFF1 (Figure 2B). Counter action of PMA and PD98059 on MEK/ERK activation was shown to result in the opposite regulation of DMBT1 transcripts, which strongly supports a direct role for ERK signaling in controlling the expression of DMBT1 in AGS cells, and possibly in the gastric progenitor cells they may represent. Furthermore, the fact that PD98059 alone increases DMBT1 expression demonstrates that not only PMA-induced, but also the basal DMBT1 expression, may be negatively regulated by ERK1/2.

G0/G1 arrest of AGS cells is accompanied by cell growth reduction, induction of cyclin D3 and p21 (Cip/Waf1) and the increase of DMBT1 expressing cells
Induction of DMBT1 expression in confluent-seeding AGS cells, prompted us to further study whether the inhibition of ERK activation would influence cell growth. Figure 3A demonstrated that PD98059 gradually arrested AGS cells into G0/G1 phase, which started at 8 h after the treatment and reached 31 ± 6% (n = 3) above the control level by 24 h. This was accompanied by an increase to 52 ± 7.5% (n = 3) reduction of relative cell number in S phase (data not shown). These findings are in agreement with a marked (68 ± 6%, n = 5) reduction of cell division detected 24 h after PD98059 treatment (Figure 3B). They are also consistent with a previous report, showing that deactivation of ERK could be sufficient for stable cell-cycle arrest into G0/G1 phase (20,21).



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Fig. 3. Effects of PD98059 on cell growth in AGS cells. (A) Flow cytometric analysis showed that PD98059 (50 µM) induced a gradual G0/G1 arrest, compared with the control level and the transient increase in the percentage of G0/G1 cells caused by serum starvation. Results are means ± SE of three separate experiments. *Significantly different from control at P < 0.05. (B) Representitive histogram shows a 60% less reduction of CFSE fluorescent value in AGS cells, after 24 h treatment by PD98059 (50 µM) relative to control cells. See online Supplementary material for a color version of this figure.

 
In addition, two major cell cycle regulators, cyclin D3 and p21 were quantified after immunocytochemical staining of AGS cells. PD98059 elicited an early increase of p21 positive cells, which lasted for at least another 8 h (Figure 4A). This observation is in line with the report that the induction of p21 could be related to ERK deactivation (22), which is known to be responsible for G1 arrest (21). Meanwhile, PD98059 induced cyclin D3 at 8 h, which was an average of 2.5-fold (n = 3) above the control level by 12 h (Figure 4A). In addition to the promotion of G1–S transition (21,23), cyclin D3 has also been reported to be upregulated in response to differentiation stimuli, when it blocks G1 progression through its binding to pRb (23).



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Fig. 4. PD98059 induction of the expressions of G1-phase regulators and DMBT1 in AGS cells. (A) PD98059 (50 µM) increased the percentage of cyclin D3 and p21 (Cip/Waf1) positive cells, which was revealed by immunostaining profiles (a, original magnification, x100); and quantification (b) using a grid-counting system (values are means ± SE of three separate experiments). (B) Treatment with PD98059 for 24 h increased DMBT1 synthesizing cells ~3 fold (b), compared with basal level (a). Original magnification, x100. Values are mean ± SE of three independent experiments. See online Supplementary material for a color version of this figure.

 
Finally, DMBT1 expression accompanying these cellular events was quantified after immunostaining. An ~3-fold increase (n = 5) of AGS positive cells was found after 24 h of PD98059 administration (Figure 4B).

Induction of DMBT1 expression in AGS cells seeded at high density, accompanied by the de-activation of ERK and the reduction of cell growth
A remarkable induction of DMBT1 at both mRNA (Figure 5A) and protein (Figure 5B) levels was detected from super-confluent cells compared with subconfluent ones. Notably, DMBT1 positivity was only present in the subpopulation of cells that reached confluence (Figure 5B, b). Stronger staining was detected in super-confluent areas (Figure 5B, c), which was absent before cells reached confluence (Figure 5B, a).



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Fig. 5. Induction of DMBT1 expression coincides with the reduction of cell growth and ERK activity, in AGS cells seeded at high density (confluent and super-confluent). (A) RT–PCR analysis revealed the induction of transcripts of both DMBT1 and the growth reduction marker GKLF, in super-confluent seeded (H) AGS cells, based upon the intensity of signals in subconfluently seeded (L) cells. (B) Immunostaining showed the absence of DMBT1 positive cells in subconfluent cells (a), and the presence of such cells in confluent (b) and super-confluent (c) cells. Original magnification, x100. (C) ERK phosphorylation was determined in proteins extracted from subconfluent (L), confluent (C) and PD98059 (PD, 50 µM) treated AGS cells.

 
GKLF/KLF4 is an epithelia-specific transcription factor known as an inhibitor of cell cycle, whose induction is associated with growth arrest in gastrointestinal epithelial cells (24). Upregulation of GKLF transcripts was seen in high-density cells 6–40 h post seeding, relative to its level in low-density cells (Figure 5B). Its induction therefore marks the reduction of AGS proliferation. The result was not influenced by the depletion of serum, which suggests that the change in growth observed here is caused by an intrinsic program induced by high-seeding density, rather than a cellular response to external growth factors.

Confluent-seeding, similar to PD98059 treatment, has also been reported to negatively regulate the activity of MAP kinase/ERK signaling in intestinal epithelial cells (20). Similarly, we demonstrated that ERK activity seen in subconfluent cells was significantly decreased in confluent cells (Figure 5C).

Silencing of DMBT1 has no significant effect on the expression of GKLF and TFF1
To further study the role of DMBT1 in the process of gastric epithelial differentiation, we used siRNA directed against DMBT1 to reduce the induction of its expression in post-confluent AGS cells. To control for non-specific effects induced by transfection of siRNA oligonucleotides into AGS cells, we tested the effects of a mismatch siRNA duplex in parallel. RT–PCR analysis showed that wild-type but not mutated siRNA, effectively decreased the gene expression of DMBT1 in post-confluent cells (Figure 6). Induction of GKLF, the marker for cell growth arrest, in post-confluent AGS cells, was not inhibited by exposure to DMBT1 siRNA. However, compared with cells transfected with mismatch siRNA control, AGS cells transfected with the DMBT1 siRNA showed less PMA induction of the gene expression of TFF1.



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Fig. 6. Effects of DMBT1 siRNA on AGS cells. AGS cells transfected with siRNA duplex directed against DMBT1 or a mismatch control were harvested before cells reaching confluence (L), or left to reach confluence (H) and treated with PMA (50 ng/ml) for 24 h. Following treatment, RNA was extracted from the cells to determine gene expression levels of DMBT1, GKLF and TFF1. TfR specific product was used as an internal control. Representatives of three independent experiments are shown.

 
Differential expression of DMBT1 in gastric adenocarcinoma
The potential association between the expression level of DMBT1 and the initiation of gastric epithelial differentiation, predicts its abnormality in tumorigenesis. We examined this hypothesis using biopsies from patients with gastric adenocarcinoma. Normal distribution of DMBT1 was detected in the neck region of the gastric mucosa (Figure 7A, arrow head), as reported previously (11). Absence of DMBT1 was seen in 80% (8/10) samples histologically defined as diffuse-type (Figure 7A and B), which is known to be poorly differentiated and fast growing (25). Interestingly, significant induction of DMBT1 was found in all eight samples of intestinal type (Figure 7C and D), which is well differentiated and slow growing (25).



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Fig. 7. Differential expression of DMBT1 in patients with gastric adenocarcinoma. Immunohistochemical analysis was performed using a mAb against DMBT1. Although positivity was detected along the neck region in the gastric mucosa (arrow head), there was no signal of DMBT1 in transformed areas (arrows) of diffuse-type adenocarcinoma (A and B). Induction of DMBT1 was detected in intestinal-type cancer (C and D, open arrows), relative to the intensity seen in the gastric mucosa (A).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
DMBT1 has been related to carcinogenesis since it was first cloned at a genomic level (1). Initial studies showed its deletion and loss of expression in a variety of tumors, indicating its potential as a tumor supressor (36). However, recent reports revealed a more complicated profile of its deletion, polymorphism and expression in these cancers (4,26,27). Thus, the particular role of this gene in tumorigenesis remains unclear.

To answer this question, we focus on analyzing the possible physiological function of DMBT1 using a gastric cell line AGS, in which the change in DMBT1 level has been related to the process of cell differentiation (11). Using specific inhibitors to explore the same cell system, we identified the involvement of two intracellular signal molecules PKC and MEK/ERK, in mediating the regulation of DMBT1 expression. In particular, the downstream location of ERK along signal transduction pathways allows it to converge signals from various extracellular stimuli, leading to the direct initiation of an array of cellular events. These include proliferation in response to mitogens (2831), and differentiation in vivo (31) and in vitro of different cell lines (20,22,32,33), depending possibly on the intensity/duration of ERK activation (20,30) or the cell-cycle properties (31). Manipulation of this signal molecule rather than PKC, the kinase lying proximal to the cell membrane, may provide more reliable association between DMBT1 expression and specific cellular responses.

Indeed, we demonstrated that in AGS cells, through inhibiting ERK activation, PD98059 blocked both PMA downregulation of DMBT1, and its induction of cell cycle withdrawal and the tissue-specific expression, events which represent epithelial differentiation. In line with a previous report showing that PD98059 interferes with the differentiation-specific expression in the intestinal cell line Caco-2/15 (20), our results implicate that transient ERK activation was required in mediating both gastric epithelia differentiation and the expression of DMBT1, which strongly suggest an association between the two events.

A switch from cell proliferation to terminal differentiation is not only fundamental in the development of organisms, but also in the frequent regeneration of certain tissues in adulthood under physiological and pathological conditions, whose abnormalities are closely related to tumorigenesis. This process consists of sequential cellular events in gastrointestinal epithelia, where the G0/G1 arrest, exit from the cell cycle/cease of cell growth and the onset of differentiation-specific expression proceed in order. They precisely coincide with alternations of the relevant signal molecules, such as PKC and ERK subfamily of mitogen-activated protein kinases (20,21,34). Two distinct stages can be defined by the states of ERK activation, as well as coordinated cellular events, as exemplified in the intestinal cell line Caco2–15: decrease in ERK activity is associated with G1 cell cycle block, followed by its re-activation, which leads to the expression of differentiation specific markers (20,22). Sequential cellular behavior and corresponding changes in ERK activation have also been reported in the in vitro differentiation processes in skeletal muscle cells (32,35), neuronal cells (36) and HL-60 cells (22). These studies suggest that alternations in ERK signaling effectively promote the stepwise transition from cell proliferation to differentiation in a wide variety of cell types.

On one hand, our results showed the PMA induction of cell-cycle exit and the expression of tissue-specific genes, phenotypes that apparently fit into the second stage of differentiation when PMA activates ERK kinase, as seen in other cell systems (22,3739). Gradual downregulation of DMBT1 expression was observed during this time period.

On the other hand, the inhibition of ERK activation by PD98059, arrested an increasing number of AGS cells into G1 phase within 24 h without the induction of TFF1 mRNA. Consistent with this finding, PD98059 was found to induce a portion of cells to express cyclin D3 and p21, both of which have been regarded as the major mechanisms to drive cell differentiation by conducting G1 arrest (23,40). Noticeably, time-course studies revealed that PD98059 induction of DMBT1 transcripts became recognizable, only after its inhibition of ERK phosphorylation in AGS cells and driving ~15% of cells into G1 phase (Figure 3A). It is believed that differentiation is initiated within a prolonged G1 phase, in which cells would commit to terminal differentiation (23). These sequential changes in AGS cells thus led to the speculation that only those cells in G1 phase with a potential to commit to differentiate, would start producing DMBT1. Moreover, it is believed that only a small percentage of cells arrested in the prolonged G1 phase will be targeted by various reagents to undergo differentiation [normally ~15%, (23)], which may well explain the low percentage of AGS cells that were DMBT1 positive in either PD98059-treated (Figure 4B), or confluently seeded (Figure 5B) AGS cells.

This notion was supported by the induction of DMBT1 in high-density seeded AGS cells, which also showed a cell growth reduction within 24 h without the initiation of differentiation expressions. With the decrease in ERK activity, 24 h post-confluent culture may well represent the initial stage of differentiation, which was always the culture background used for PMA addition. Furthermore, studies using siRNA directed against DMBT1 also suggested that DMBT1 may have no effect on cell growth arrest, but may influence the differentiation-specific expression.

It is worth noticing that Wu et al. (41) and Takeshita et al. (6) reported a substantial downregulation of DMBT1 expression in lung cancer cell lines, whereas Peterson et al. (4) did not confirm these observations even from the same cell lines (4). Reminiscent of the results reported by the group of Al-Awqati (8), the present study demonstrated that DMBT1 expression largely depended on the cell culture conditions, particularly on the cell density. We further showed that culture conditions are reflected in different levels of activities of signal molecules, which in turn may directly regulate the expression of DMBT1. These observations may help dissolve the controversial discussion on DMBT1 expression in cancer cell lines.

Taken together, studies using these complementary culture conditions and gene silencing suggest a particular stage for DMBT1 expression in gastric epithelia: it is turned on early in differentiation-committed cells, during their accumulation into prolonged G1 phase, and turned off after these cells begin to display the differentiation phenotypes and exit cell cycle (Figure 8).



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Fig. 8. Schematic model of the association between expression levels of DMBT1 and different stages during the proliferation to differentiation switching process in AGS cells, defined by the collective usage of various biological parameters. The activation of ERK is represented by the blocks in green and yellow; the suppression of ERK activation is represented by the blocks in mauve.

 
Human DMBT1 is primarily located within the proliferating compartment of the gastric and intestinal mucosa, coinciding with the distribution of stem/progenitor cells (3). However, DMBT1 may be present only in those cells that are destined to undergo differentiation during the migration upward or downward along the mucosa axis (20,42) because this molecule was merely detectable either in actively proliferating subconfluent AGS cells that have no tendency to differentiate, or in terminally differentiated superficial gastric epithelia (11). These observations collectively indicate a tight temporal and spatial control of the expression of DMBT1 in vivo, which has been well reflected in AGS cells upon various treatments. Such an expression pattern, right between the proliferation to differentiation switch, suggests an important role for DMBT1 in the cell fate decision, particularly along the gastrointestinal tract, with yet unknown mechanisms. This view fits well into a recent report, in which a highly inducible expression of a rat DMBT1 was found in transit-amplifying ductular (oval) cells (possibly originating from endogenous stem cells) in regenerating rat liver (43). The close association between the expression level of DMBT1 and the initiation of gastric epithelial differentiation is consistent with its absence in the majority of poorly differentiated diffuse-type adenocarcinoma, and its induction in the better differentiated intestinal-type. These observations suggest that although DMBT1 is involved in gastric epithelial differentiation, it may not be a decisive factor for oncogenesis, which is primarily a disorder of cell proliferation, with different degree of abnormalities in differentiation. The difference in its expression levels in cancer areas may reflect the difference in the degree of cell differentiation among malignant tissues. Therefore, the usefulness of DMBT1 as a good biomarker to help predict the prognosis of gastric cancer is worth further investigation.


    Supplementary material
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Supplementary material can be found at: http://www.carcin.oupjournals.org.


    Acknowledgments
 
We appreciate the skillful technical assistance from Peter Ottosen and Raymond Daniels. We also thank Dr Siamon Gordon and Dr Jan Mollenhauer for their critical reading of the manuscript.

Conflict of Interest Statement: None declared.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 

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Received October 2, 2004; revised January 25, 2005; accepted February 6, 2005.





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