Epstein-Barr virus induces human nasopharyngeal epithelial cells to escape from the replicative senescence

Objective. To observe the biological changes of primary human nasopharyngeal epithelial cells in the early stage of immortalization. Methods. The morphological changes of nasopharyngeal epithelial cells were observed by phase-contrast microscopy, and the activity profile of senescence-associated β-galactosidase (SA-β-Gal) was detected by SA-β-Gal staining. The expression of p16^INK4a protein was tested by immunochemical assay, and the life span in vitro of nasopharyngeal epithelial cells was calculated as population doublings. In addition, the expression of Epstein-Barr (EB) virus latent membrane protein 1 (LMP1) was also detected by immunofluorescence staining. Results. Morphologically, cells treated with EB virus and 12-o-tetradecanoyl-phorbol-13-acetate (TPA) formed multi-layer foci, and their cellular life span in vitro was extended (about 155 days of culture). A low percentage of cells (about 4.8%) expressed SA-β-Gal activity at late primary culture, and did not always express p16^INK4a protein in the progression of culture. Conclusions. Nasopharyngeal epithelial cells treated with EB virus in cooperation with TPA can pass through the stage of senescence and enter the early stage of immortalization. Some changes of phenotype occur in these cells. Our results provide data for further studying the mechanism of immortalization and the establishment of a human nasopharyngeal epithelial cell line.