236 Gut 2001;49:236–240 Overexpression of the CD155 gene in human colorectal carcinoma Gut: first published as 10.1136/gut.49.2.236 on 1 August 2001. Downloaded from D Masson, A Jarry, B Baury, P Blanchardie, C Laboisse, P Lustenberger, M G Denis Abstract monoclonal antibodies (E4) reacts strongly Background and aims—The Ta ge 4 gene with the primary tumours and lung metastases, (tumour associated glycoprotein E4) is as determined by immunohistochemistry. The overexpressed in rat colon tumours and epithelia of the gastrointestinal tract and of the Min mouse intestinal adenomas. The rat bronchi are only slightly stained and no Tage4 protein has approximately 40% staining of other normal rat tissues is observed. identity with human CD155, a member of Initial experiments have indicated that the the immunoglobulin superfamily coding antigen recognised by E4 is a transmembrane for a transmembrane protein capable of glycoprotein.23The corresponding Ta g e 4 gene serving as an entry receptor for polio- (tumour associated glycoprotein E4) has since virus, porcine pseudorabies virus, and been found to be overexpressed in chemically bovine herpesvirus 1. Analysis of the rat induced rat colon tumours4 and in Min mouse Ta ge 4 gene has revealed structural and intestinal adenomas.5 In contrast, a barely functional similarities with the human detectable level was found in the normal adult CD155 gene. We therefore investigated rat and mouse tissues tested. The Ta g e 4 cDNA expression of the CD155 gene in human has been isolated.6 Sequence analysis using colorectal carcinomas. ALIGNp software revealed 40.9% identity Methods—Overall CD155 expression was between the Tage4 protein and the human 7 assessed by semiquantitative reverse CD155 a member of the immunoglobulin (Ig) transcription-polymerase chain reaction superfamily coding for a transmembrane pro- (RT-PCR) and immunohistochemical tein capable of serving as an entry receptor for analysis using tissue specimens from poliovirus, porcine pseudorabies virus, and patients with colorectal adenomas and bovine herpesvirus 1. The Ta g e 4 gene has been 8 adenocarcinomas. We also used a qualita- mapped to rat chromosome 1q22 and mouse 9 tive RT-PCR assay to determine relative 7A2-B1, regions that are homologous to the expression of diVerent splicing variants in long arm of human chromosome 19 where the 10 each sample. CD155 gene is located. To gain insight into Results—mRNA levels of CD155 were the molecular mechanisms involved in its over- http://gut.bmj.com/ expression in cancer cells, we isolated and increased in six of six colorectal cancer 11 tissues compared with the tumour free characterised the rat Ta g e 4 gene. This gene ∼ colon mucosa. Immunohistochemical covers 15 kb and is composed of eight exons analysis revealed an increased level of and seven introns. The human CD155 gene CD155 protein in 12 of 12 samples. The also contains seven introns in identical posi- qualitative RT-PCR assay revealed that tions and phases. In addition, the protein relative expression of the diVerent CD155 encoded by the Ta g e 4 gene was tested for abil- on October 2, 2021 by guest. Protected copyright. variant transcripts was similar in the ity to mediate entry of several viruses. The diVerent normal and cancer samples results indicated that the Ta g e 4 gene is tested, indicating that this overexpression probably orthologous to the gene for CD155. is not associated with a particular mRNA We therefore decided to analyse expression of Laboratoire de variant generated by alternative splicing the CD155 gene in human colorectal carci- Biochimie Médicale, of the CD155 gene. noma (CRC). This was performed by reverse Faculté de Médecine, transcription-polymerase chain reaction (RT- 44035 Nantes, France Conclusion—We have shown for the first time that the CD155 gene is overexpressed PCR) analysis and by immunohistochemistry. D Masson We also analysed relative expression of the dif- B Baury in colorectal carcinoma and that this P Blanchardie overexpression begins at an early stage in ferent variants of the CD155 gene by a qualita- P Lustenberger tumorigenesis and continues to late tive RT-PCR assay. M G Denis stages. (Gut 2001;49:236–240) Materials and methods INSERM U539, Faculté TISSUE SAMPLES de Médecine, 44035 Keywords: CD155; colorectal cancer; immunoglobulin Tissue samples were obtained from 18 patients Nantes, France superfamily; poliovirus receptor AJarry (10 men and eight women; mean age 66 years, C Laboisse range 45–90) undergoing surgical resections for primary CRC diagnosed at the Department Correspondence to: As our understanding of the biology of of Surgery, Nantes University Hospital, Dr MG Denis, Laboratoire colorectal cancer progresses, new knowledge de Biochimie Spécialisée, France. Adenomas were collected from four Institut de Biologie, 9 quai about tumorigenesis and tumour biology can patients: two villous adenoma (both high grade Moncousu, F-44035 Nantes be used to diagnose, treat, and prevent this type Cedex, France. of cancer. In an initial study of an animal model [email protected] Abbreviations used in this paper: CRC, colorectal for immunotherapy of colon tumours, mono- carcinoma; RT-PCR,reverse transcription-polymerase Accepted for publication clonal antibodies directed against rat colon chain reaction; CAM, cellular adhesion molecule; 29 January 2001 carcinoma cells were raised.1 One of these CEA, carcinoembryonic antigen; Ig, immunoglobulin. www.gutjnl.com CD155 and colorectal cancer 237 dysplasia) and two tubular adenomas (one low heated at 72°C for three minutes and then grade and one high grade dysplasia). Sporadic cooled on ice. It was combined with 0.5 µg of tumours were collected from 14 patients and random hexamers (Promega, Lyon, France), classified according to Astler and Coller: stage transcription buVer (50 mM Tris HCl, pH 8.3, Gut: first published as 10.1136/gut.49.2.236 on 1 August 2001. Downloaded from B (n=4), stage C (n=7), and stage D (n=3). 75 mM KCl, 3 mM MgCl2, 10 mM DTT), The tumours were localised in the right colon dNTPs (1 mM each), RNasin (50 units; (n=5), transverse colon (n=2), left colon Promega), and RNaseH− M-MLV reverse (n=3), sigmoid (n=2), and rectum (n=2). transcriptase (200 units; Promega) in a final Thirteen tumours presented a moderate diVer- volume of 20 µl. Incubation was performed at entiation, and one was highly diVerentiated. 42°C for 60 minutes. From each patient tumour tissue and adjacent normal mucosa (about 10 cm from the SEMIQUANTITATIVE EXPRESSION OF THE CD155 tumour) were excised and collected. Samples GENE were immediately processed for isolation of Amplifications were performed with 3 µl of epithelial cells and fragments were frozen for cDNA in a total volume of 50 µl containing 10 immunohistochemistry and kept at −70°C mM Tris HCl, pH 9.0, 50 mM KCl, 1.5 mM until analysis. MgCl2, 0.1% Triton X-100, 0.2 mg/ml gelatin, 200 µM of each dNTP, 100 pmol of specific NORMAL COLONIC EPITHELIAL CELLS AND primers, and 2.5 units of Taq DNA polymerase CANCER CELL LINES (Promega). Normal epithelial cells were isolated from sur- Primers were designed from the published gical specimens using a non-enzymatic disso- sequence of the human CD155 gene.10 They ciation technique based on the use of the were designed to amplify cDNA but not calcium chelating agent EDTA, as previously contaminating genomic DNA. The primers described.12 used to quantify expression of the CD155 gene HCT8R, SW620, and SW1116 colon carci- were located in a region corresponding to the noma cell lines were grown in RPMI-1640 extracellular domain of the protein, which is containing L-glutamine and sodium bicarbo- conserved in all splice variants (fig 1). The nate (Sigma, Saint Quentin Fallavier, France) sequences of these primers were: CD1–5' supplemented with 10% fetal calf serum, peni- primer: 5'-TATCTGGCTCCGAGTGCTT cillin (100 units/ml), and streptomycin (0.1 GCC-3'; CD2–3' primer: 5'-ATCATAGCCA mg/ml). Cells were maintained at 37°Cin5% GAGATGGATACC-3'. carbon dioxide and passaged twice a week. For semiquantitative amplification, each cycle was carried out at 92°C for 30 seconds, IMMUNOHISTOCHEMISTRY 62°C for 30 seconds, and 72°C for 60 seconds. Cryostat sections (5 µm) were fixed in acetone The reaction was stopped after 18 cycles, in the and stored at −20°C. Before use, slides were linear range of the amplification (not shown). http://gut.bmj.com/ rehydrated with phosphate buVered saline (150 Following electrophoresis, amplified DNA was mM NaCl, 10 mM phosphate buVer, pH 7.4). transferred to Hybond N+ membranes (Amer- The slides were then incubated with normal sham Pharmacia Biotech, Orsay, France). The goat serum for 10 minutes. Mouse monoclonal nylon filter was hybridised using alkaline phos- antibody D171 (Neomarkers, Union City, phatase labelled probes using the AlkPhos sys- California, USA) directed against human tem (Amersham Pharmacia Biotech). For CD15513 was used as a primary antibody at a CD155, a 362 bp insert amplified by PCR concentration of 2.5 µg/ml in antibody diluent from HCT8R cell cDNA with oligonucleotides on October 2, 2021 by guest. Protected copyright. containing background reducing components CD1 and CD2 was used as a probe. (Dako, Trappes, France). Alkaline phosphatase Normalisation was performed by RT-PCR goat antimouse Ig (Sigma) was used as a amplification and hybridisation of â-actin secondary antibody (dilution 1:50). Following cDNA. Fourteen cycles (92°C for 30 seconds, two washes with phosphate buVered saline and 58°C for 30 seconds, 72°C for 30 seconds) one with Tris buVered saline (150 mM NaCl, were performed with oligonucleotides B1 10 mM Tris Cl, pH 7.5), staining was (5'-CCTTCCTGGGCATGGAGTCCT-3') developed using the Vector Red alkaline phos- and B2 (5'-GGAGCAATGAT-CTTGATCT phatase substrate (Vector, Burlingame, Califor- TC-3').