SUMMARY:A membrane invasion culture system was used to study the ability of EBV to enhance invasion and migration of nasopharyngeal carcinoma (NPC) cells. Semi-reverse transcriptase-PCR analysis of matrix proteinases and angiogenic factors from EBV-infected, or EBV-positive (EBVϩ), cells demonstrated different degrees of elevated gene expression. In our animal model, EBVϩ tumors grew faster and larger than EBV-free, or EBV-negative (EBVϪ), tumors and also had clonal EBV terminal repeat sequences. Double-localization of EBV and certain host proteins in EBVϩ tumors and biopsy specimens demonstrated that EBV up-regulates host genes only in cells that express those genes but not in cells that do not express them. Double-localization of EBV and host genes in NPC biopsy specimens all showed EBVϪ tumor cells expressing those host genes. Our data strongly suggest that EBV infection enhances progression of NPC tumor growth. They do not rule out a role for EBV infection in the induction and early promotion of NPC development. Unidentified factors may also enhance NPC tumor growth independent of the effects of EBV. (Lab Invest 2003, 83:797-812).

To understand whether the p53-regulated mdm2 gene expression was altered by the Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC), the NPC-TW01 cell line was infected by EBV through IgA receptor-mediated endocytosis. The mdm2 gene was expressed only in a small fraction of the NPC cell population and could be enhanced in the EBV-infected (EBV þ ) cells. In the animals bearing EBV þ and EBV À NPC xenografts, the MDM2 þ cells only appeared in clusters in both EBV þ and EBV À tumors with stronger expression in EBV þ cells. Cotransfection of pmdm2-Luc plus pSV40-p53 plus pCMV-LMP1 in the NPC-TW06 line that had p53 heterozygous point mutation showed stronger mdm2 promoter activity than cells cotransfected with pmdm2-Luc plus pSV40-p53, but no mdm2 promoter activity was seen in cells cotransfected with pmdm2-Luc plus pCMV-LMP1. Only the EBV-LMP1 but not the EBV-LMP2A gene could enhance p53 to upregulated mdm2 expression. Tumor cells in NPC biopsy specimens revealed similar mdm2 expression as in the animal model. It is concluded that although EBV can indirectly enhance mdm2 gene expression in tumor cells that express this gene, it cannot turn on or directly regulate mdm2 expression in cells that do not express this gene. In other words, EBV plays a role as an enhancer in NPC tumorigenesis.