MATERIALS AND METHODS

Cell culture

The gastric cancer cell lines MKN28, KATO III, and AGS, and the colon cancer cells Colo 320 and Colo 205 were obtained from the American Type Culture Collection, and were routinely cultured in RPMI-1640 (Life Technologies, Paisley, UK) with 10% (vol/vol) fetal calf serum (Sera Lab, Crawley, Surrey, UK) supplemented with 5mM glutamine and 40 μg/ml gentamicin.

Patients

Gastric mucosal biopsy samples were obtained from patients undergoing routine upper gastrointestinal endoscopy. Informed consent was obtained from each patient, and the study was approved by the local clinical research ethics committee. Patients who had received antisecretory agents, antimicrobial treatment, or non-steroidal anti-inflammatory drugs in the preceding two months were excluded from our study. Biopsies samples were immediately snap frozen in liquid nitrogen and stored at −80°C for subsequent extraction of RNA. A rapid urease test (CLO test; Delta West, Australia) was performed on an additional antral biopsy sample. Samples were also taken for histological assessment by the Sydney system.¹² Patients were determined to be H pylori infected if positive by the CLO test, histological assessment, or reverse transcription polymerase chain reaction (RT-PCR) for ureA. Gastric tumour tissue was also obtained from eight patients undergoing surgery for gastric cancer and immediately frozen at −80°C. Histological analysis showed six of the gastric cancers were of the intestinal type, one was diffuse type, and one mixed intestinal and diffuse types.

RNA extraction and RT-PCR analysis

RNA was extracted from gastric and colon cancer cell lines and from the gastric tumour samples and biopsies by means of a cationic detergent based extraction method (Catrimox-14; Iowa Biotechnology, Iowa, USA).¹³ Extracted RNA samples were treated with 1 unit of DNase I (Life Technologies) and then reverse transcribed as described previously.¹³ cDNA was amplified by PCR with primers specific for zFOC1 and the house keeping gene, glyceraldehyde 3 phosphate dehydrogenase (GAPDH) (table 1). Each PCR contained 0.5 pmole of oligonucleotide primers in a total volume of 20 μl. Thermal cycling conditions were as follows: predenaturation at 95°C for five minutes, denaturation at 95°C for one minute, annealing at 55°C for one minute, and extension at 72°C for one minute. cDNA was amplified for 35 cycles for GAPDH and 40 cycles for zFOC1. PCR was also carried out with an RNA sample to confirm the absence of contaminating genomic DNA. After amplification, PCR products were separated by 1% (wt/vol) agarose gel electrophoresis and visualised by ultraviolet illumination .

Northern hybridisation

A Multiple Tissue Northern Blot (Human Digestive System; BD Clontech, Basingstoke, Hampshire, UK), for which 1 μg polyA⁺ RNA from 12 different digestive system tissues had been size fractionated before transfer to a nylon membrane, was hybridised to a [³²P] radiolabelled zFOC1 probe. This probe was prepared by the random primer method (Life Technologies) using the purified 349 bp PCR product described in table 1. Hybridisation was performed at 68°C according to the manufacturer’s instructions. The blot was washed three times at 50°C in wash solution 1 (2× saline sodium citrate (SSC) and 0.05% sodium dodecyl sulfate (SDS) (wt/vol)) and twice in wash solution II (2× SSC and 0.1% SDS (wt/vol)). It was then exposed to x ray film at −70°C.

RESULTS

Sequence analysis of zFOC1

zFOC1 is an evolutionary well conserved protein. Human and guinea pig zFOC1 show 99% homology at the amino acid level (fig 1). A comparison of zFOC transcript sequences found on the DNA databases (accession numbers AL834266 and AK056034) with the genomic sequence from bacterial artificial chromosome clone AC068790 indicated that this gene has a rather unusual structure (table 2). All introns lie within the 5′UTR. A comparison between zFOC mRNA sequences from accession numbers AL834266 and AK056034 indicates that they share the same length of 3′UTR. The sequence that encodes zFOC1 and the 3′UTR accounts for 3275 bp of the AL834266 sequence. Alternative splicing apparently begins at position 1231. This indicates that the largest exon, exon 7, is the 3′UTR exon of 3875 bp (table 2). Northern blot analysis indicated the presence of a transcript size of approximately 4.3 kb in the poly A⁺ mRNA purified from all gastrointestinal tissues examined (fig 2). These data suggest that the additional 500 bp of the 5′UTR is made up of a combination of some of the much smaller exons described in table 2.

Expression of zFOC1 in digestive tissues and gastrointestinal cancer cell lines

A Multiple Tissue Northern Blot of poly A⁺ RNA extracted from various normal gastrointestinal tissues was screened to study the distribution of zFOC1 transcripts (fig 2). The highest expression was seen in liver, followed by duodenum and stomach (fig 2). In contrast, much lower transcript levels were found in the oesophagus, rectum, and caecum (fig 2).

Previous cDNA array analysis studies indicated that zFOC1 transcripts were present in the gastric epithelial cell line KATO III.¹⁰ This observation was confirmed by RT-PCR analysis. zFOC1 transcripts were found in total RNA extracted from the gastric cancer cell lines KATO III, AGS, and MKN28, and the colon cancer cell lines, Colo 205 and Colo 320 (fig 3A; lanes 12–16).

Increased expression of zFOC1 transcripts in total RNA from gastric carcinomas than in normal gastric mucosa and H pylori infected gastric mucosa

RT-PCR was used to screen eight gastric tumour samples for the presence of zFOC1 transcripts. They were identified in six of the eight samples (fig 3B; lanes 1–4, 7, 8). The two tumour samples in which zFOC1 transcripts were not identified were both intestinal-type gastric cancers. Using these same conditions of PCR analysis, zFOC1 transcripts could not be detected in most of the total RNA samples prepared from histologically normal gastric mucosa (corpus and antrum) of patients with or without H pylori infection (fig 3A). zFOC1 expression was seen in the antrum of only one of the nine patients with H pylori infection (fig 3A; lane 5), and in none of the antral biopsies from 13 uninfected controls (for example, fig 3A; lanes 6–11). The expression of transcripts was much lower in this sample than in the cell lines run in parallel (fig 3A; compare the band intensities seen in lanes 12–16 with that in lane 5). Biopsies from the corpus were also screened for the expression of zFOC1. Of the five patients who were positive for H pylori, tissue from one patient weakly expressed the transcript. The corpus mucosa of the patients who were negative for H pylori (n = 5) did not show the presence of zFOC1 transcripts.

DISCUSSION

In a previous study, the expression of zFOC1 was found to be increased in the KATO III gastric cancer cell line following stimulation with H pylori.¹⁰ We have now established that zFOC1 transcripts are also detectable by northern blot analysis in polyA⁺ RNA purified from a range of gastrointestinal tissues. However, much higher expression was found in gastric tumour samples and gastric cancer cell lines when compared with histologically normal gastric mucosa, and in mucosal samples from H pylori infected patients with chronic gastritis. Although our in vitro cDNA array data indicated that zFOC1 expression was increased in gastric cancer cells after coculture with H pylori,¹⁰ the data presented above suggest that a similar increase is unlikely to be occurring in non-neoplastic gastric epithelial cells of H pylori infected patients. The higher expression of zFOC1 in gastric cancer tissue implies that this gene may be functionally important in tumour development.

zfOC1 was originally cloned from the guinea pig organ of Corti.¹¹ The protein is characterised by the presence of nine potential zinc finger motifs. Zinc fingers have been found in many transcription factors, including Sp1^14–16 and TFIIIA,¹⁷ and steroid and thyroid receptors.¹⁸ These motifs are generally associated with the DNA binding domain activity of the protein. Progressive deletion of zinc finger repeats in TFIIIA results in a parallel loss of DNA binding activity.¹⁹

“The higher expression of zFOC1 in gastric cancer tissue implies that this gene may be functionally important in tumour development”

Helicobacter pylori infection is associated with a pronounced increase in gastric epithelial cell proliferation.^20,21 The molecular mechanism that regulates this process has not been established. Several transcription factors have been implicated in malignant cell growth, and these factors can regulate growth, survival, invasiveness, and changes in gene expression.^22,23 For example, the transcription factor Ets-1 is thought to have a role as a predictor of metastases and as a prognostic marker in patients with gastric cancer.²⁴ As a consequence, therapeutic strategies have been implemented with the aim of inhibiting the activity of transcription factors, in an attempt to reduce cancer cell proliferation and invasiveness.²⁵ Thus, reducing the activity of zFOC1 may also be considered as a feasible therapeutic strategy.

It remains to be clarified whether the upregulation of zFOC1 is related to H pylori infection or whether it is a marker of gastric cancer in general. A larger screen including patients with and without infection and premalignant lesions would be informative. In fact, although transcription factors such as nuclear factor κB are activated in gastric epithelial cells by cag PAI positive H pylori strains in vitro and by bacterial infection in vivo,^26,27 other factors have not been extensively investigated. It would also be of interest to identify those target genes regulated by zFOC1 in gastric cancer cells. In conclusion, zFOC1 is differentially expressed in normal and malignant gastric epithelial cells. The functional role of zFOC1 in gastric cancer requires further investigation.