Isolation and cultivation of colon epithelial cells (CEC) were performed as previously described[8,9].

The colon carcinoma cell lines SW48 (ATCC CCL-231), SW480 (ATCC CCL-228), CaCo-2 (ATCC HTB-37), LoVo (ATCC CCL-229), HCT116 (ATCC CCL-247), and HT29 (ATCC HTB-38) were used for in vitro experiments. The cell line HT29M3 was chosen as a highly differentiated colon carcinoma cell line[10-13].

For tissue culture, the cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with penicillin (400 U/mL), streptomycin (50 µg/mL), L-glutamine (300 µg/mL), and 100 mL/L fetal calf serum (FCS) (Sigma, Deisenhofen, Germany) and split 1∶5 every three days.

For demethylation assays, the cells were treated with 5-azacytidine at a final concentration of 10 µmol/L for 48 h (Sigma)[8]. HT29 M3 cells were used as control cells in the demethylation experiments.

For RT-PCR, total cellular RNA was isolated from cultured cells or laser microdissected tissue samples using the RNeasy kit (Qiagen, Hilden, Germany). The integrity of the RNA was controlled on 10 g/L agarose/formaldehyde gel, and subsequently cDNAs were generated by reverse transcriptase reactions. The reverse transcription (RT) reaction was performed in 20 µL reaction volume containing 2 µg of total cellular RNA, 4 µL of 5x first strand buffer (Gibco), 2 µL of 0.1 mol/L DTT, 1 µL of dN₆ primer (10 mmol/L), 1 µL of dNTPs (10 mmol/L) and DEPC water. The reaction mix was incubated for 10 min at 70 °C. Then 1 µL of Superscript II reverse transcriptase (Gibco) was added and RNAs were reverse transcribed for 1 h at 37 °C. Subsequently, reverse transcriptase was inactivated at 70 °C for 10 min and RNA was degraded by digestion with 1 µL of RNase A (10 mg/mL) at 37 °C for 30 min. cDNAs were controlled by PCR amplification of ß-actin.

The entire coding region of Chibby was amplified by RT-PCR from cDNA using16, ATGCCTTTCTTTGGGAATTACGTTC (forward) and chibby496, TCATTTTCTCTTCCGGCTGATC (reverse), which resulted in a 380-bp fragment. The PCR reaction was performed in 50 µL reaction volume containing 5 µL of 10x Taq buffer, 1 µL of cDNA, 0.5 µL of each primer (0.2 µmol/L), 0.5 µL of dNTPs (10 mmol/L), 0.5 µL of Taq polymerase (5 U/µL), and 41 µL of water. The amplification reactions were performed by 35 repetitive cycles of denaturation for 30 s at 94 °C, annealing for 30 s at 64 °C, extension for 30 s at 72 °C, and a final extension step at 72 °C for 5 min. The PCR products were resolved on 15 g/L agarose gels. For sequencing, the products were purified by PEG precipitation to remove unincorporated primers and dNTPs. Both strands were sequenced for each PCR product from at least two independent PCR reactions. Sequences were compared with the gene data bank by means of BLAST search (National Center of Biotechnology Information).

Quantitative real-time PCR was performed on a LightCycler (Roche, Mannheim, Germany). Two microliters of cDNA template, 1.6 µL of 25 mmol/L MgCl₂, 0.2 µmol/L forward and reverse primers {chibby125, TTTGGGAATACGTTCAGTCCG (forward) and chibby395, TCAGCCGCAAGAGATTGTTC (reverse)} and 2 µL of SybrGreen LightCycler mix in 20 µL volume were PCR-amplified through 40 cycles, each cycle consisting of initial denaturation at 95 °C for 30 s, 20 °C/s temperature transition rate up to 95 °C for 15 s, 64 °C for 3 s, 72 °C for 5s, 85 °C acquisition mode single. The PCR product was evaluated by melting curve analysis following the manufacturer’s instructions and by checking the PCR products on 18 g/L agarose gels. All quantitative PCR experiments were repeated three times.

A total of 2×10⁵ cells were seeded onto six-well plates one day before transfection. Transfections with pCMX PL2 or pCMX PL2 flag Chibby plasmid (0.5 µg per well) were performed using the LipofectAMINE plus method (Life Technologies) according to the manufacturer’s instructions. Cells were harvested 48 h after transfection and the amount of Chibby expression was determined by RT-PCR and Western blot analysis.

For protein isolation, 2×10⁶ cells were washed in 1x PBS and lysed in 200 µL RIPA buffer (Roche). The protein concentration was determined using the BCA protein assay reagent (Pierce, USA). Equal amounts of cellular protein (40 µg total protein) were denatured at 94 °C for 10 min after addition of Rotiload buffer (Roth, Karlsruhe, Germany) and subsequently, separated on NuPage SDS gels (Invitrogen, Groningen, The Netherlands). After transferring the proteins onto PVDF membranes (BioRad, Richmond, USA), the membranes were blocked in 50 g/L dry milk in TBS-T and incubated overnight with primary monoclonal anti flag antibody (1∶4 500 dilution, Sigma) at 4 °C. A 1∶4 000 dilution of anti IgG AP (Sigma) was used as secondary antibody. Staining was performed using 5-bromo-4-chloro-3-indolyl phosphate/ nitroblue tetrazolium tablets (Sigma). All Western blot experiments were repeated at least three times.

A total of 2×10⁴ SW480 cells were seeded per chamber of four-well-Falcon chamber slides (Becton Dickinson, Le Pont de Plaix, France) and cultivated overnight before transfection with either control vector or Chibby flag pCMX PL2. After 48 h of transfection, cells were washed with PBS, fixed with 40 g/L paraformaldehyde, permeabelised with 1 ml/L Triton X-100, washed again, and blocked for 30 min in 10 g/L BSA in PBS. Anti flag antibody (dilution 1∶1 000) was used as primary antibody, and anti mouse FITC-conjugated antibody (dilution 1∶40) was used as secondary antibody. Incubations took place at room temperature for 1 h each. Staining of nuclei occurred with DAPI.

For transient transfections, 2×10⁵ cells per well were seeded into six-well plates and transfected with 0.5 µg of TOPflash or FOPflash reporter gene plasmids using the LipofectAMINE plus method (Gibco) according to the manufacturer’s instructions. For cotransfection, 0.125 µg, 0.25 µg, 0.5 µg, and 1 µg of Chibby expression plasmid per well were used, respectively. At 48 h post-transfection, the cells were lysed and the luciferase activity in the lysate was measured. To normalise transfection efficiency, 0.2 µg of a pRL-TK plasmid (Promega, Mannheim, Germany) was cotransfected and renilla luciferase activity was measured by a luminometric assay (Promega). All transfection experiments were repeated at least three times.

Proliferation was measured as previously described[14]. Proliferation of Chibby transfected SW480 cells was compared to non or mock transfected SW480 cells in an XTT assay. Results were expressed as mean ± SD (range). All experiments were repeated three times.

Invasion assays were performed as previouly described[14]. Briefly, the assays were performed in Boyden chambers containing polycarbonate filters with an 8-µm pore size. In brief, filters were coated with a commercially available reconstituted basement membrane (Matrigel; Becton Dickinson, Heidelberg, Germany), and the lower compartment was filled with fibroblast-conditioned medium as chemoattractant. SW480 cells and Chibby transfected SW480 cells were harvested by trypsinization, resuspended in DMEM without FCS at a density of 2×10⁵ cells/mL, and placed in the upper compartment of the chambers. After incubation for 4 h at 37 °C, the filters were removed. The cells adhering to the lower surface were fixed, stained, and counted. Fifteen random fields were counted at 200-fold magnification. Each sample was assayed in triplicate, and all experiments were repeated three times.

We analysed a panel of seven colon carcinoma cell lines, such as SW48, SW480, CaCo2, LoVo, HCT116, HT29, and HT29 M3, that are frequently used as model systems to study effects in colorectal carcinoma. We evaluated the panel of colon carcinoma cell lines for alterations in the Chibby mRNA expression. The complete Chibby coding region was amplified by RT-PCR. All cell lines examined expressed Chibby mRNA at the expected length, indicating that no deletions or mutational insertions had occurred (Figure 1). As a control the Chibby coding sequence was amplified using CEC cDNA as template. The same cDNAs were used to amplify β-actin as a control for the integrity of the cDNA.

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Figure 1 PCR amplification of the complete coding region of Chibby mRNA. The Chibby coding region was amplified using RT-PCR. All colon carcinoma cell lines were shown to express full-length Chibby mRNA. Expression levels cannot be estimated from this experiment as the PCR was carried out to saturation in order to illustrate full-length mRNA expression and to obtain enough PCR products for sequencing. β-actin amplification served as control to ascertain the integrity of the cDNA in all samples.

The cell lines were evaluated for levels of Chibby mRNA expression using quantitative RT-PCR in comparison to normal colon epithelial cells (CEC). Chibby mRNA was found to be down-regulated in all cell lines in comparison to CEC and HT29 M3 cells which are considered to be a highly differentiated colon carcinoma cells and express Chibby in amounts similar to those of CEC (Figure 2, black bars). Three of the analysed cell lines, SW48, SW480, and CaCo2 cells, showed residual expressions of Chibby mRNA of 18%, 37%, and 35%, respectively, in comparison to CEC or HT29 M3 (100% and 106%, respectively). LoVo, HCT116, and HT29 cells were also shown to have reduced Chibby mRNA levels, but down-regulation was not as explicit as in the other examined cell lines (57%, 62%, and 46%).

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Figure 2 Quantification of Chibby expression in colon carcinoma cell lines. The amount of Chibby mRNA expression was carefully quantified by real-time PCR. All colon carcinoma cell lines showed down-regulation of Chibby mRNA expression in comparison to colon epithelial cells (CEC) and HT29 M3 cells, a cell line derived from HT29 cells that is considered to be a highly differentiated colon carcinoma cell line (black bars). Treatment of cells with the demethylating agent 5-azacytidine caused a modest increase in Chibby mRNA levels in SW48, SW480, and CaCo2 cells, but not in the other cell lines (grey bars). HT29 M3 cells were used as controls for the demethylation experiments as the experiment could not have been performed with primary CEC due to culture conditions.

To investigate the reason for down-regulation of Chibby expression in colon carcinoma cell lines, we amplified and analysed the exons of the chibby gene. All PCR products were purified and subsequently sequenced using two different primers. The analysis of sequenced PCR products did not reveal any sequence variation within the coding region of the chibby gene locus (data not shown).

From these results, we concluded that Chibby does not harbour mutations within the coding sequence in all seven analysed colon carcinoma cell lines.

As this reduction in mRNA expression in colon carcinoma cell lines could not be explained by loss or mutation of the chibby gene, we hypothesised that promoter hypermethylation could have silenced gene expression. To test this hypothesis, all cell lines were exposed to the demethylating agent 5-azacytidine. Treated and untreated cells were tested for Chibby mRNA expression which was quantified by quantitative RT-PCR. HT29 M3 cells were used as controls in these experiments. HT29 M3 cells are highly differentiated cells that were used as an additional control as CEC could not be used in demethylation experiments as controls due to culturing issues.

Our results showed that 5-azacytidine treatment did not lead to induction of Chibby expression in LoVo, HCT116, HT29, or HT29 M3 cells, but to a moderately increased expression in SW48, SW480, and CaCo2 cells (Figure 2, grey bars). Therefore, we conclude that promoter hypermethylation might play a minor role for altered Chibby expression in some of the analysed cell lines, but we do not consider this silencing mechanism to be a general event in down-regulation of Chibby mRNA expression in the colon carcinoma cell lines.

As colon carcinoma cell lines display reduced levels of Chibby expression in comparison to normal CEC, we were interested in whether over-expression of Chibby in these cells influences functional processes. To test for the potential effects, we over-expressed Chibby in the colon carcinoma cell line SW480 by transfecting a flag-tagged Chibby expression construct. Successful over-expression was observed by quantitative RT-PCR (data not shown) and Western blotting (Figure 3A). Exogenous Chibby protein was not detected following mock or control transfection with pCMX PL2 flag plasmid which did not alter Chibby levels. Strong over-expression of Chibby was observed after transfection of SW480 cells with Chibby flag expression plasmid. Immunofluorescence staining revealed correct expression patterns of Chibby flag-tagged protein in the nucleus of SW480 cells (Figure 3B), also displaying high transfection efficiency.

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Figure 3 Expression of Chibby in transfected SW480 cells. A: Western blot analysis showing Chibby protein expression levels in untransfected (mock), control vector, or Chibby transfected SW480 cells. B: immunofluorescence staining of flag-tagged Chibby transfected SW480 cells. Nuclei were stained with DAPI in blue, flag-tagged Chibby was detected with FITC-labelled anti-flag antibody (green). C: TOPflash reporter gene assays showing inhibition of β-catenin signalling after transfection of SW480 cells with Chibby expression plasmid.

As Chibby as endogenous antagonist of β-catenin is expected to exert negative functions on the Wnt/β-catenin signalling pathway, we performed LEF/TCF luciferase reporter gene assays using the TOPflash/FOPflash system. Analysing the activity of β-catenin in control and Chibby over-expressing cells, we observed reduced activation levels in the TOPflash system in accordance to the amount of transfected Chibby expression plasmid (Figure 3C). Transfection with 0.125 µg or 0.25 µg of Chibby plasmid per well did not affect the activity of TOPflash reporter, whereas activity dropped to 44% and 48%, respectively, when 0.5 µg and 1 µg expression plasmids were applied. FOPflash reporter construct activity was not affected by Chibby expression (Figure 3C). These results showed that transfection of SW480 cells with Chibby expression plasmid reveals functional Chibby protein.

To assay whether altered Chibby levels had an impact on proliferation and invasion potential of SW480 cells, we performed functional experiments utilizing mock and Chibby transfected cells. Boyden chamber assays were performed to examine the effect of Chibby on the invasiveness of SW480 cells. In this assay, the tumor cells have to invade through matrigel mimicking the basal lamina. Comparing mock treated cells, control transfected cells, and Chibby over-expressing cells, we did not observe any differences with regard to their invasiveness.

As the Wnt/β-catenin signalling pathway is known to influence cell proliferation, we next assayed for alterations of proliferation and performed in vitro proliferation assays with Chibby transfected SW480 cells and mock or control transfected cells. Modifying the Chibby protein level had no significant effect on cell proliferation (Figure 4). Doubling times were as follows: SW480: 21.2 h, mock 1: 21.7 h, mock 2: 19.7 h, cby 1: 22.6 h, and cby 2: 22.5 h, respectively. Thus, we did not consider proliferation to be affected by Chibby expression levels in SW480 cells.

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Figure 4 Proliferation Assay. Analysis of the cells over-expressing Chibby could not altered doubling time in comparison to mock or control transfected SW480 cells. Assays were performed in triplicates.

As our experiments in colon carcinoma cell lines had revealed sound down-regulation of Chibby expression, the absence of effects on cell functions of over-expressed functional Chibby protein in one of these cell lines (SW480) was rather unexpected. We next performed experiments with samples from patients with diagnosed colorectal carcinoma to assess the question whether Chibby expression was altered not only in colon carcinoma cell lines, but also in coloreactal tumors and might therefore contribute to or support development and progression of colorectal tumors.

We analysed Chibby transcript expression levels in tumor samples and corresponding normal colonical tissue from the same patients by hybridisation of an oligonucleotide-based array (Affymetrix HG-U133A) with 43 samples of colorectal cancer and 34 matched normal mucosa (unpublished data). The ratio of expression levels between tumors and normal tissues was 1.11, displaying no altered Chibby expression in CRC tumors in comparison to adjacent normal colon tissue. In addition, when tumor samples with microsatellite stability (MSS; n = 24) and microsatellite instability (MSI; n = 19) were separately assessed, again no difference in Chibby expression was observed.

To further validate this result, using quantitative RT-PCR, we tested whether expression levels of Chibby mRNA differed between normal colorectal tissue and adjacent cancerous tissue. We manually microdissected frozen material and analysed the extracted mRNAs. Figure 5 shows the results of 3 examined matched samples, displaying no profound alterations between tumor and adjacent normal tissue. Relative Chibby levels ranged from 100% in normal tissue to 117.4% (patient 1), 146.1% (patient 2), and 116.4% (patient 3), respectively.

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Figure 5 Chibby mRNA expression levels in colorectal tumor and corresponding normal tissue samples. Quantitative RT-PCR showing Chibby mRNA expression levels in matched pairs of tumor and normal colorectal tissue of 3 different patients.