Epstein-Barr virus promotes epithelial cell growth in the absence of EBNA2 and LMP1 expression

Abstract

We attempted to infect primary gastric epithelia (PGE) with recombinant Epstein-Barr virus (EBV) carrying a selectable marker that made it possible to select EBV-infected cells. Cells dually positive for EBV-determined nuclear antigen (EBNA) and cytokeratin were detected in 3 of 21 primary cultures after 3 days of EBV inoculation. From one culture, EBV-infected cell clones were repeatedly obtained at a frequency of 3 to 5 cell clones per 10(6) cells. EBV-infected clones had enhanced population doubling and grew to attain a highly increased saturation density, together with acquisition of marked anchorage independence. The infected clones retained the ultrastructural morphology characteristic of gastric mucosal epithelium and have been growing stably for more than 18 months (corresponding to at least 300 generations) so far, in clear contrast to the parental PGE cells, which ceased growth after 60 generations. The p53 gene of the parental PGE cells was found to be overexpressed, perhaps thereby conferring the basal potential for long-term survival in vitro. Moreover, EBV infection accelerated, to a significant extent, the growth rate and agar clonability of NU-GC-3 cells, an established EBV-negative but EBV-susceptible human gastric carcinoma cell line. Both EBV-converted PGE and NU-GC-3 clones, like EBV-positive gastric carcinoma biopsy specimens, expressed a restricted set of EBV latent infection genes characterized by the absence of EBNA2 and latent membrane protein 1 (LMP1) expression. These results indicate that EBV infection causes a transformed phenotype on PGE in the setting of possible unregulated cell cycling and renders even established gastric carcinoma cells more malignant via a limited spectrum of viral latent-gene expression. This study may reflect an in vivo scenario illustrating multiphasic involvement of EBV in carcinogenesis of gastric or other epithelial cancers.

FIG. 1

Detection of EBV in G418-resistant PGE-5 clones. (A) Immunofluorescent staining of EBNA in a G418-resistant PGE-5 clone with EBV-seropositive human serum. Magnification, ×400. (B) Immunofluorescent staining in the same PGE-5 clone with EBV-seronegative human serum as a control. 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 DNAs served as positive controls. Each lane of PGE-5 clones and parental PGE-5 cells contained 5 μg of DNA. All G418-resistant PGE-5 clones were estimated to carry more than 25 copies of the EBV genome per cell.

FIG. 2

Demonstration of the epithelial nature of PGE-5 cells. (A) Immunoblot analysis for cytokeratin expression in EBV-infected and -uninfected PGE-5 cells. A mixture of MAbs AE1 and AE3 detected cytokeratins as multisized specific bands between 40 and 66 kDa. A gastric carcinoma cell line, NU-GC-3, served as a positive control, and LCL served as a negative control. (B and C) Transmission electron micrographs of EBV-infected PGE-5 cells show the junctional complex (arrows) (B) and mucus granules (arrows) (C). Bars in panels B and C denote 200 nm and 1 μm, respectively. Magnifications, ×8,000 (B) and ×20,000 (C).

FIG. 3

Analysis of EBV latent-gene expression in EBV-infected PGE-5 cell clones. (A) Immunoblotting for detection of EBNAs and LMP1. The blots were probed with pooled human sera for EBNA1, EBNA2, EBNA3A, EBNA3B, and EBNA3C (top blot), MAb PE2 for EBNA2 (middle blot), and MAb CS1-4 for LMP1 (bottom blot). Protein samples extracted from 10⁵ cells were loaded per slot. (B) In situ hybridization for EBER1. An EBV-infected PGE-5 clone was hybridized with the antisense oligoprobe (left) and with the sense oligoprobe (right). Intense nuclear signals are evident only with the antisense probe. (C) RT-PCR analysis of EBV latent-gene expression and EBNA promoter usage in EBV-infected clones. Akata cells were used as a positive control for detection of Qp-initiated EBNA mRNA, and LCL was used as a positive control for detection of LMP1, LMP2A, LMP2B, and BARF0 mRNAs and Cp- or Wp-initiated EBNA mRNAs. Parental PGE-5 cells served as a negative control.

FIG. 4

Growth characteristics of Neo^r-transfected and EBV-infected PGE-5 clones. (A) Neo^r-transfected PGE-5 clone. Magnification, ×100. (B) EBV-infected PGE-5 clone. Magnification, ×100. Both clones were detached by trypsinization, seeded into separate wells of 12-well plates under the same culture conditions, and photographed at near the plateau phase (5 days after passage). Differences between the two cell types are easily recognizable (see the text for details). (C) Growth kinetics of EBV-infected (solid circles) and Neo^r-transfected (open circles) PGE-5 cells at normal and low FCS concentrations. Individual data are plotted as a circle with a vertical bar, which represents the mean ± standard error calculated from the results of four clones.

FIG. 5

Growth in soft agarose of EBV-infected PGE-5 clones and controls. (A) Neo^r-transfected PGE-5 clone as a control. Magnification, ×40. (B) EBV-infected PGE-5 clone. Magnification, ×40. A total of 10⁴ cells at the logarithmic phase were seeded per well in six-well plates. The number of viable colonies was counted and photographed 4 weeks after seeding. Almost all colonies in the control culture consisted of dead cells, whereas the virus-infected clone formed many large colonies.

FIG. 6

Immunoblot analysis of p53 expression in EBV-infected and -uninfected PGE-5 cells. The gastric carcinoma cell line NU-GC-3 was used as a positive control for p53 overexpression. PGE-17 and PGE-21 denote other PGE lysates.

FIG. 7

(A) Detection of EBNAs (top blot) and LMP1 (bottom blot) in EBV-converted NU-GC-3 cells by immunoblotting. (B) EBV induced-growth alteration of NU-GC-3 cells. Growth curves of EBV-infected (solid circles) and Neo^r-transfected NU-GC-3 (open circles) clones in medium containing 10% FCS are shown. The assay was carried out in six-well culture plates. Data are expressed as described in the legend to Fig. 4C.