Infection of breast epithelial cells with Epstein-Barr virus via cell-to-cell contact.

Epstein-Barr virus (EBV), a human herpesvirus present in more than 90% of adults, is a major viral cofactor in certain tumors of lymphoid and epithelial tissues (1). Persistent infection is associated with malignancies and proliferative syndromes typically of lymphoid and epithelial tissues (1), including Burkitt’s lymphoma, Hodgkin’s disease, certain adult T-cell lymphomas, and, in epithelium, nasopharyngeal carcinoma and oral hairy leukoplakia. In vitro, EBV efficiently infects, transforms, and immortalizes B cells, yielding lymphoblastoid cell lines (LCLs). Several studies have associated EBV with breast cancer. Bonnet et al. (2) detected EBV genomes and gene expression in breast cancer lesions by using polymerase chain reaction (PCR) analysis, Southern hybridization, and immunohistochemistry specific for EBV protein EBNA (i.e., EBV nuclear antigen)-1. Labrecque et al. (3) detected EBV in breast cancers by PCR and in situ hybridization. There are descriptions of EBV-associated lymphomas (4,5) localizing to breast and of bilateral breast cancer developing during the rare chronic active EBV infection syndrome (6). Recent reports (7,8) have described EBV infection of human carcinoma cells on cocultivation with LCLs by a mechanism requiring cell-to-cell contact. These findings and the reported association with breast cancer prompted us to address the question of whether EBV enters breast epithelium by cell-tocell contact. We have developed an appropriate reagent: EBV bearing the gene encoding and expressing the protein known as enhanced green fluorescent protein (EGFP) (9,10). Cells infected by this virus, designated EBfaV-GFP, are readily detected by their green fluorescence (9–11). Here, we report that cells of human breast cancer epithelial lines T47D and MCF7, which are not infected on direct exposure to cell-free EBfaV-GFP virus, become infected when cocultivated with LCLs derived with and bearing EBfaVGFP, as shown by expression of EGFP. This finding is consistent with a possible role for EBV in the etiology of breast cancer. EBfaV-GFP, with EGFP driven by a strong promoter, within a dispensable region of the viral genome is produced as described previously (10). MCF7 and T47D cells (derived from human breast tumors) and Daudi cells (an LCL immortalized by and bearing wild-type