Epstein-Barr virus and gastric carcinoma

Abstract

The Epstein-Barr virus (EBV) is detected in the tissue of about 10% of gastric carcinoma cases throughout the world. In each case, 100% of carcinoma cells are infected with EBV. Analysis of EBV in carcinoma biopsies indicates that carcinoma is formed by the proliferation of a single EBV infected cell. These findings suggest that EBV plays an important role in the development of EBV positive gastric carcinomas. The EBV genes expressed are EBV determined nuclear antigen 1 (EBNA1), two small non-polyadenylated RNAs known as EBER1 and EBER2, and the transcripts from the BamHI-A region (BARF0); in addition, some cases also express a small amount of latent membrane protein 2A (LMP2A). Epithelial cells are refractory to EBV infection in vitro. This has hampered the study of the role of EBV in epithelial malignancies. The use of recombinant EBV carrying a selectable marker has enabled this difficulty to be overcome. EBV infected cell clones can be obtained from most carcinoma cell lines examined, and it was found that cell to cell contact was an efficient mode of EBV infection. Furthermore, it was possible to immortalize primary gastric epithelial cells by EBV infection. The cells expressed identical EBV genes to those typically seen in EBV positive gastric carcinoma, and showed accelerated malignant properties, including growth in soft agarose and tumorigenicity in severe combined immunodeficient (SCID) mice. These results suggest that EBV contributes to the maintenance of the malignant phenotype of EBV positive gastric carcinoma.

Figure 1

Detection of Epstein-Barr virus (EBV) encoded small RNA 1 (EBER1) in gastric carcinoma of the differentiated type. All carcinoma cells are positive for EBER1.

Figure 2

World distribution of Epstein-Barr virus (EBV) positive gastric carcinoma.

Figure 3

Epstein-Barr virus (EBV) DNA detection in EBV positive gastric carcinoma biopsies by Southern blotting. The EBV DNA terminal repeat (TR) was used as a probe. A single band was detected for each gastric carcinoma tissue, indicating that the gastric carcinoma was formed by proliferation of one EBV infected cell. Lane 1, EBV producing cell line B95-8 (multiple bands because the number of viral TRs is different for each virus particle); lane 2, Burkitt's lymphoma cell line Raji (single band); lanes 3–19, EBV positive gastric carcinoma samples (single bands); lane 20, EBV negative gastric carcinoma. Kindly provided by Dr S Imai, Kochi Medical School, Kochi, Japan.

Figure 4

Detection of Epstein-Barr virus (EBV) in G418 resistant PGE-5 clones. (A) Immunofluorescent staining of EBV determined nuclear antigen (EBNA) in a G418 resistant PGE-5 clone with EBV seropositive human serum. Original magnification, ×400. (B) Immunofluorescent staining of the same clone with EBV seronegative human serum as a control. Original magnification, ×400. (C) Southern blot analysis of G418 resistant PGE-5 clones. All DNA samples were digested with BamHI and the blot was probed with a BamHI-K fragment of EBV DNA. Serially diluted Raji cell DNA served as positive controls. Each lane contained 5 μg of DNA. All G418 resistant PGE-5 clones were estimated to carry more than 25 copies of the EBV genome/cell. Reproduced from Nishikawa et al.

Figure 5

Growth characteristics of neomycin resistance gene (Neo^r) transfected and Epstein-Barr virus (EBV) infected PGE-5 clones. (A) Neo^r transfected PGE-5 clone. Original magnification, ×100. (B) EBV infected PGE-5 clone. Original magnification, ×100. Both clones were detached by trypsinisation, seeded into separate wells of 12 well plates under the same culture conditions, and photographed near the plateau phase (five days after passage). Differences between the two cell types are easily recognisable. (C) Growth kinetics of EBV infected (closed circle) and Neo^r transfected (open circle) PGE-5 cells at normal (10%; left) and low (0.1%; right) fetal calf serum (FCS) concentrations. Each point represents the mean ±SE of the results for four clones. Reproduced from Nishikawa et al.