Clone-specific expression, transcriptional regulation, and action of interleukin-6 in human colon carcinoma cells

From the Caco-2 cell line (ATCC HTB-37), which was originally derived from a well differentiated human colon adenocarcinoma, a number of homogenous and stable Caco-2 clones were established in several laboratories. For the present study, we used the clone Caco-2/AQ, which was derived from the Caco-2/15 clonal line [16] as described before [17]. Caco-2/AQ cells undergo spontaneous differentiation on transition into the post-confluent state, as indicated by a steep rise in differentiation markers, i.e. alkaline phosphatase activity (cf. Table 1).

Primary colon adenocarcinoma cell clones COGA-1 and COGA-13 were isolated by Drs. Ernst Wagner and Alexandra Sinski at Boehringer Ingelheim Austria, Vienna, as described in detail elsewhere [18]. From the COGA-1 clonal cell line, which was derived from a Dukes' stage B, pT3, moderately differentiated (i.e. G2) carcinoma, a morphologically homogenous sub-clone, designated COGA-1A, was established and characterised with respect to growth behaviour and degree of differentiation, i.e., alkaline phosphatase activity (Table 1; cf. [5]).

COGA-13 cells, which were derived from a stage pT2 carcinoma graded G3, exhibit only weak alkaline phosphatase activity (Table 1). In addition to the epithelial cell marker cytokeratin 8 (CK8), COGA-13 cells express also a high level of vimentin [19]; this indicates that they had undergone epithelial-mesenchymal transition.

Human colon cancer cells were routinely cultured in vented tissue culture flasks (Asahi Techno Glass Corporation, Iwaki Scitech division, Tokyo, Japan) at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Culture medium was DMEM supplemented with 4.0 mM glutamine, 10% (v/v) fetal calf serum (FCS) (heat-inactivated at 56°C for 30 min), 20 mM HEPES, 50 U/ml penicillin and 50 μg/ml streptomycin. Cultures were re-fed every 48 h and subcultured serially when approximately 80% confluent. Cells between passages 6 and 24 were cultured for indicated time periods in the absence or presence of one of the following treatments: 5 ng/ml IL-1β (ImmunoTools, Friesoythe, Germany); 10^-6 M indomethacin, 10^-7 M PGE₂, 10^-7 M 17β-estradiol, 10^-6 M NS398 (all from Sigma), and 10^-8 M 1,25-(OH)₂D₃ (a generous gift from Hoffmann-La Roche, Basle, Switzerland).

The effect of 0.1 – 100 ng/ml recombinant human (rh) IL-6 (Strathmann Biotec AG, Hamburg, Germany) on cellular proliferation was determined by measurement of [³H]thymidine incorporation into cellular DNA. Degree of cellular differentiation was evaluated in confluent cells from activity of the marker enzyme alkaline phosphatase as described in detail previously [20]: Enzymatic activity was assayed using p-nitrophenol as substrate, and normalised to cellular protein content, which was determined using the BCA Protein Assay Kit (Pierce, Rockford, IL).

Total RNA was extracted by using Trizol (GibcoBRL). 2 μg RNA were reverse transcribed with random hexamer primers using a cDNA synthesis kit (SuperScript™ II, Invitrogen). First-strand cDNA was amplified with primer pairs for IL-6, and for the reference gene and epithelial cell marker CK8, respectively (MWG-Biotech AG, Ebersberg, Germany). To amplify a 349 base pair segment of IL-6 cDNA, primer pairs used were 5'-TTC-AAT-GAG-GAG-ACT-TGC-CTG-3' (sense) and 5'-ACA-ACA-ACA-ATC-TGA-GGT-GCC-3' (antisense) [21]. The primer pairs for c-myc were 5'-GGC-TTT-ATC-TAA-CTC-GCT-GT-3' (sense) and 5'-GAG-GTC-ATA-GTT-CCT-GTT-GG-3' (antisense) to amplify a 461 base pair segment [22]. Primer pairs for a 520 base pair segment of the CK8 gene were 5'-TGG-GCA-GCA-GCA-TTA-ACT-TTC-3' (sense) and 5'-AGG-CGA-GAC-TCC-AGC-TCT-AC-3' (antisense) [23]. With the aid of the GeneAmp PCR System 9600 (Perkin-Elmer, Norwalk, CT), the thermocycling conditions began with 94°C for 2 min followed by 34 cycles (IL-6) and 30 cycles (CK8), respectively, of: 94°C 15 sec, 62°C 30 sec, 72°C 45 sec, or followed by 30 cycles (c-myc) of 94°C 30 sec, 57°C 60 sec, 72°C 60 sec. Each run was concluded by 10 min at 72°C. PCR conditions and primer pairs for cDNA amplification of CYP27B1, CYP24, and VDR were previously provided elsewhere [19], as well as for cDNA amplification of ER-α and ER-β [24]. A 1:1 mixture of PCR products and CK8 amplificates from the same sample was separated on 2% agarose gels and then visualised with ethidium bromide. No bands were visible in negative controls that were performed for all primer pairs.

IL-6 concentrations in cell culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA) (eBioscience, San Diego, CA) according to the protocol of the manufacturer. Since preliminary data had indicated that some clones might produce IL-6 in amounts close to the detection limit of the assay (2 pg/ml IL-6), we added a constant background of 10 pg/ml IL-6 to all samples to improve intra-assay accuracy. Immunoblotting for CK8, vimentin, cyclin D1, and p27 has been described elsewhere [19].

Human genomic DNA was extracted from confluent colorectal cell clones. Cells were trypsinised and washed twice with PBS before suspension in extraction buffer (50 mM Tris / HCl pH 8, 100 mM EDTA, 100 mM NaCl, 1% SDS, 0.5 mg/ml proteinase K) and overnight shaking on an Eppendorf shaker at 55°C. Saturated NaCl (6 M) (a third of the volume of extraction buffer) was added before centrifugation at 13000 rpm for 10 min on a Microfuge Lite Centrifuge (Beckman Coulter, Fullerton, CA). The supernatant was treated with approximately the same amount of isopropanol, shaken thoroughly, and centrifuged (13000 rpm, 5 min). Thereafter, the pellet was washed with EtOH (70%, v/v), resuspended in 400 μl TE buffer (10 mM Tris, pH 8 / 1 mM EDTA), and agitated on an Eppendorf shaker at 65°C for 15 min. DNA was stored at -20°C until analysed. DNA amplification was performed with a GeneAmp PCR System 9600 (Perkin-Elmer, Foster City, CA) as described previously [25]. PCR products were subsequently purified using the Centri Sep 96 system (Princeton Separations, NJ), and thereafter subjected to sequencing using Big Dye Terminator Cycling Sequencing kit, version v3.1, and the ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA) according to the manufacturer's recommendation. Primers for amplification and sequencing were applied as listed in [9].

Statistical analyses were conducted with use of the software packages S-PLUS (version 4.5, Lucent Technologies Inc., Murray Hill, NJ) and SPSS (version 12.0.1, SPSS Inc., Chicago, IL). The normal distribution of all data sets was verified with the aid of the one-sample Kolmogorov-Smirnov goodness of fit-test; as a result the unpaired Student's t-test was used throughout. With a confidence level of 0.95, differences were considered statistically significant with *, P < 0.05; **, P < 0.01; ***, P < 0.001.

We had availed ourselves of three human colon carcinoma-derived cell clones, i.e. Caco-2, COGA-1A, and COGA-13, which we had characterised previously with respect to their log phase growth behaviour and to expression of the differentiation marker alkaline phosphatase [5] (Table 1). For the purpose of the present study, the proliferative potential was evaluated by determination of the cell cycle regulators, cyclin D1, which promotes G1/S transition, and p27, a cdk-inhibitor. We also determined expression of the epithelial and mesenchymal cell markers, cytokeratin 8 (CK8) and vimentin, respectively. Furthermore, we checked for expression of the vitamin D metabolising enzymes, 25-hydroxy-vitamin D-24-hydroxylase (CYP24) and 25-hydroxy-vitamin D-1α-hydroxylase (CYP27B1), as well as of the vitamin D receptor (VDR), and the estrogen receptors (ER)-alpha and -beta (Table 1).

Consistent with their origin from a well differentiated tumour, Caco-2 cells exhibited the highest level of the differentiation marker alkaline phosphatase. In contrast, enzyme activity was low in COGA-1, and was barely detectable in COGA-13 cells. Cellular differentiation declined in the order Caco-2 > COGA-1A > COGA-13. The proliferative potential varied inversely with differentiation, as indicated by the rise in cyclin D1 and the drop in p27 (Table 1).

All three cell clones were of epithelial origin according to their expression of the epithelial cell marker CK8 (Table 1). Little vimentin was detected in Caco-2 and COGA-1A cells, whereas in COGA-13, expression of vimentin exceeded that of CK8. This indicates a high degree of epithelial/mesenchymal transition in these least differentiated cancer cells (Table 1).

The findings listed in Table 1 also indicate that all three clones, notwithstanding marked differences in growth characteristics and degree of differentiation, express the VDR, and, as typical for colon epithelial cells, the ER-β [24]. Consequently, all three clones can be expected to respond to the receptor ligands, i.e., 1,25-(OH)₂D₃ and 17β-E₂. Also, all three clones expressed the vitamin D metabolising enzymes, 25-hydoxyvitamin D-24-hydroxylase (CYP24) and 25-hydoxyvitamin D-1α-hydroxylase (CYP27B1), though at markedly different levels. Of note, CYP27B1 was abundant in Caco-2/AQ cells, as is typical for rather differentiated colon carcinomas, whereas CYP24 was overexpressed in COGA-13 cells consistent with a low degree of differentiation [26].

RT-PCR analysis revealed a distinct, i.e., clone-specific, pattern of basal and stimulated expression of IL-6 mRNA in human colon carcinoma cells (Figure 1): As had been observed previously, carcinoma cell clones derived from well to moderately differentiated cancers, i.e. Caco-2 and COGA-1A, expressed only small amounts of IL-6, as compared with COGA-13 cells, which are derived from a poorly differentiated tumour. In these cells, which had already undergone substantial epithelial-mesenchymal transition, IL-6 is conspicuously overexpressed.

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In Caco-2/AQ cells, IL-6 gene activity and protein synthesis were up-regulated only by IL-1β, whereas COGA-1A responded only to 1,25-(OH)₂D₃. In COGA-13 cells, IL-6 levels were increased to some extent by PGE₂, whereas IL-1β increased IL-6 expression by three orders of magnitude (Figure 1).

A more detailed picture of clonal sensitivity towards modulators of IL-6 was obtained when IL-1β, PGE₂, 1,25-(OH)₂D₃, and 17β-E₂, singly or in appropriate combinations, were added to colon carcinoma cell cultures (Table 2). Overexpression of IL-6 by the COGA-13 clone became apparent also at the protein level, as COGA-13 cells produced at least four times more IL-6 than Caco-2/AQ or COGA-1A cells (Table 2). In none of the clones, IL-6 production, regardless of whether basal or IL-1β-induced, was changed when endogenous prostaglandin synthesis was blocked by indomethacin (Table 2) or by the cyclooxygenase (COX)-2 inhibitor, NS398 (not shown). As mentioned before, when 10^-7 M PGE₂ was added to COGA-13 cultures, a small increment in IL-6 release was observed.

17β-E₂ was effective in COGA-13 cultures, where it reduced basal IL-6 release by one third. However, the hormone did not alter the extensive stimulation by IL-1β of IL-6 synthesis (Table 2).

We analysed the IL-6 gene promoter for the presence of specific polymorphisms that are known to influence transcriptional regulation of IL-6, e.g., by IL-1 and PGE₂ [9] (Table 3).

To evaluate the mitogenic potency of IL-6 in Caco-2/AQ, COGA-1A and COGA-13 cell cultures, graded concentrations of the cytokine (0–100 ng/ml) were added to the medium for 72 h. A dose dependent increase in cell division was observed only in Caco-2/AQ cells, whereas the growth rate in COGA-1A and GOGA-13 was unaltered (Figure 2A). All three clones are endowed with the IL-6 receptor [5], but IL-6 proliferative signaling was transduced to c-myc expression only in Caco-2 cells (Figure 2B): IL-6 expression rose about fourfold after 24 h incubation with 100 ng/ml rhIL-6 (Figure 2C).

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In Caco-2 cell cultures, 10^-8 M 1,25-(OH)₂D₃ abolished the growth stimulatory effect of 10 ng/ml IL-6. Still a 50% reduction was observed when cell division was stimulated by IL-6 at the supraphysiological concentration of 100 ng/ml (Figure 3). 1,25-(OH)₂D₃ completely prevented any inhibitory effect of IL-6 on the activity of the differentiation marker enzyme, alkaline phosphatase (Figure 4).

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