Affiliation of authors: MicrobiologyImmunology Department, Northwestern University Medical School, Chicago, IL.
Correspondence to: Richard Longnecker, Ph.D., MicrobiologyImmunology Department, Northwestern University Medical School, Ward 6-231, 303 East Chicago Ave., Chicago, IL 60611 (e-mail: r-longnecker{at}nwu.edu).
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-to-cell 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 (911).
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 EBfaV-GFP, 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 EBV) were obtained from the American Type Culture Collection (ATCC), Manassas, VA, and were cultured according to the recommendations of the ATCC. Binding of monoclonal antibody 323/A3 (Lab Vision, Fremont, CA) against epithelial-specific antigen (ESA), abundant on the surface of T47D and MCF7 cells, and Cy5-conjugated goat-anti-mouse secondary antibody (Jackson ImmunoResearch, West Grove, PA) were measured by flow cytometry as described previously (9). Antibody 72A1 (12) against EBV glycoprotein gp350 was used in antibody-blocking experiments. Plasmid pEGFP.N1 (Clontech Laboratories, Inc., Palo Alto, CA), used in control experiments to achieve transient expression of EGFP, was transfected by electroporation into Daudi cells as described previously (10).
Monolayers of T47D or MCF7 cells in 12-well plates, at 50% confluence, were overlaid with equal numbers of GFP57 cells, which is an EGFP-positive lymphoblastoid cell line derived from primary B cells, by the method described previously (10) with the use of EBfaV-GFP virus. In control experiments, monolayers either were overlaid with equal numbers of Daudi cells transiently expressing EGFP (after adjustment for the proportion of cells expressing EGFP) or were exposed to cell-free filtered EBfaV-GFP virus produced as described previously (9) and assayed for infectious titer with the use of Daudi cells. After 24 hours of cocultivation, supernatants were removed, and cell monolayers were washed repeatedly with medium and visually examined with the use of an inverted fluorescence microscope (Leica Microscopes, Wetzlar, Germany). In monolayers cocultivated with GFP57 lymphoblastoid cells, approximately 1%3% of cells within the monolayer expressed EGFP (Fig. 1, panel 2, showing T47D cells), indicating that infection had occurred. In addition, a small number of rounded EGFP-positive cells, with morphology resembling that of LCLs, appeared to be adhering to the top of the monolayer. No EGFP-expressing cells were present within monolayers exposed to cells transiently expressing EGFP (Fig. 1
, panel 4), and again a small number of rounded EGFP-positive cells, morphologically resembling LCLs, appeared to be adhering to the top of the monolayer. Similar results were seen with MCF7 cells (data not shown).
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To address the possibility that LCLs on cultivation yielded free virus that then infected by direct virus-cell contact, cocultivations were repeated with a blocking antibody. Antibody 72A1 against EBV glycoprotein gp350, when included in cocultivations at a range of concentrations (040 µg/mL), the highest of which completely abrogates infection of Daudi cells by EBfaV-GFP virus, did not reduce the proportion of T47D or MCF7 cells that became infected (data not shown). We conclude that EBV cell-to-cell infection of these epithelial cells does not require the presence of free virus.
Efficient infection of T47D and MCF7 cells by cell-to-cell contact requires actively growing cells. Repeating cocultivation experiments with the use of completely confluent monolayers yielded cells positive for both ESA and EGFP numbering between 0.2% and 0.3% of total cells, which was two to three times the background level (data not shown).
Previous reports (7,8) have shown that EBV infects epithelial cells by cell-to-cell contact. Our observation is consistent with these findings and extends to breast epithelium the range of tissue types potentially infectible by EBV; it also supports the notion that virus-bearing lymphocytes may serve as virus donors for infection of epithelial cells (8). Cell-to-cell spread by fusion of infected cells with uninfected cells has been documented for other viruses, e.g., herpes simplex virus (13,14), pseudorabies virus (15), human immunodeficiency virus (16), and paramyxoviruses such as measles (17). Although the current studies do not elucidate the mechanism of the cell-to-cell spread of EBV, fusion of infected cells with uninfected cells is a possible explanation for this phenomenon. Alternatively, close cell-to-cell contact could augment the accessibility of virus to recipient cells, possibly with viral attachment and entry via a hypothetical low-affinity receptor molecule or molecules. Examination of the mechanism will be a subject of future experiments. EBV association with epithelial tumors has been difficult to reconcile with the apparent inability of EBV virions to efficiently undergo direct entry into epithelial cells, which express little, if any, CD21 (the major receptor for EBV). These observations begin to address the difficulties in understanding a role for EBV in breast cancer etiology by demonstrating EBV entry into breast epithelium by cell-to-cell contact.
NOTES
Supported by Public Health Service grants CA62234 and CA73507 (National Cancer Institute) and DE13127 (National Institute of Dental and Craniofacial Research) from the National Institutes of Health, Department of Health and Human Services (to R. Longnecker). R. Longnecker is a Scholar of the Leukemia Society of America.
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Manuscript received May 15, 2000; revised August 28, 2000; accepted September 5, 2000.
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