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Infection of Breast Epithelial Cells With Epstein-Barr Virus Via Cell-to-Cell Contact

Peter Speck, Richard Longnecker

Affiliation of authors: Microbiology–Immunology Department, Northwestern University Medical School, Chicago, IL.

Correspondence to: Richard Longnecker, Ph.D., Microbiology–Immunology 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. 1Go, 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. 1Go, 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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Fig. 1. Enhanced green fluorescent protein (EGFP) expression in breast epithelial cells after contact with EGFP-expressing lymphoblastoid cell lines (LCLs). Subconfluent monolayers of T47D breast epithelial cells in 12-well plates were cocultivated for 24 hours with equal numbers of GFP57 cells, an EGFP-positive LCL derived from primary human B cells, with the use of the EBfaV-GFP virus. After cocultivation, lymphoblastoid cells (which exist in suspension) were removed by washing monolayers three times with medium to remove suspended cells, and monolayers were examined by use of a Leica inverted microscope equipped for fluorescence. Panels 1 and 2 show the same field viewed by phase-contrast and fluorescent illumination, respectively. Approximately 1%–3% of cells in the monolayer expressed EGFP. In addition, a small number of rounded EGFP-positive cells, with morphology resembling that of lymphoblastoid cell lines, appeared to be adhering to the top of the monolayer. Cocultivation of T47D cells with Daudi cells induced to transiently express EGFP (panels 3 and 4, illuminated as above) by electroporation with plasmid p.EGFP.N1 did not result in EGFP expression in cells within the monolayer. Monolayer cells are negative for EGFP expression (panel 4). A small number of rounded EGFP-positive cells, with morphology resembling that of Daudi cells, appeared to be adhering to the top of the monolayer. Original magnification x400 for all panels.

 
The abundant expression of ESA by T47D and MCF7 cells is shown in Fig. 2Go (panels 1 and 2). GFP57 and Daudi cells do not express ESA (panels 3 and 4). T47D and MCF7 cells are not infected by direct contact (Fig. 2Go, panels 6 and 7) with a 100-µL inoculum of cell-free EBfaV-GFP virus that contained sufficient virus to infect 20% of 105 Daudi cells, as measured by the proportion of cells expressing EGFP (Fig. 2Go, panel 5).



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Fig. 2. Two-color flow cytometry confirms Epstein-Barr virus (EBV) infection of MCF7 and T47D breast epithelial cells by cell-to-cell contact. In two-color flow cytometry, expression of epithelial-specific antigen (ESA) serves as a marker to distinguish epithelial cells cocultivated with GFP57, an enhanced green fluorescent protein (EGFP)-expressing lymphoblastoid cell line (LCL) derived by immortalizing primary B cells with EBfaV-GFP virus. Binding of monoclonal antibody 323/A3 against ESA and Cy5-conjugated goat-anti-mouse secondary antibody was measured by flow cytometry as described previously 9. ESA expression is present in abundance on MCF7 cells (panel 1, heavy line) and T47D cells (panel 2, heavy line). ESA is not present on GFP57 (panel 3) or Daudi (panel 4) cells. Dotted lines are negative control signals produced by omission of primary antibody. MCF7 and T47D cells are not infected by direct contact with EBfaV-GFP virus: On exposure of 2.5 x 105 cells to sufficient free virus to infect 20% of Daudi cells (panel 5), neither MCF7 cells (panel 6) nor T47D cells (panel 7) become infected. Cytometer was calibrated to place uninfected (EGFP-negative) cells (on the x-axis) and ESA-negative cells (on the y-axis) within the first power of 10 on logarithmic scales spanning 4 powers of 10. Relative fluorescence intensity is depicted on an arbitrary logarithmic scale and does not have units. Abundant expression of EGFP on GFP57 cells is depicted in panel 8. Cocultivation of MCF7 or T47D cells with GFP57 cells resulted in the presence of many doubly positive cells—i.e., ESA-positive and EGFP-positive cells. Doubly positive cells are in the upper right quadrant, and the number in the box is the percentage of total cells falling in this quadrant. In the case of MCF7, after cocultivation, doubly positive cells (1.1%, panel 9) were present at a 12-fold greater number than in the control (panel 11). In T47D cells, the effect was more marked, with a 34-fold increase in the number of doubly positive cells (3.4% [panel 10] versus 0.10% [panel 12]). Panels 11 and 12 show controls, with MCF7 and T47D cells, respectively, cocultivated with Daudi cells transiently expressing EGFP, with percentages of doubly positive cells being <=0.10% of the total. The same background level of ESA-positive/EGFP-positive cells was evident when GFP57 cells were mixed with T47D cells immediately before cytometry, suggesting that the background level did not arise during cocultivation (data not shown). Differences in cell numbers in the lower right quadrants of panels 9 compared with panel 11 and panel 10 compared with panel 12 reflect the relatively tight scatter of GFP57 cells in flow cytometry, compared with the very wide scatter displayed by electroporated Daudi cells. This difference in scatter resulted in relatively fewer of the EGFP-positive Daudi cells impinging on the gate employed in flow cytometry to visualize T47D or MCF7 cells. Each cytometry plot shows at least 20 000 events.

 
To confirm that EGFP expression was occurring in breast epithelial cells in the monolayer and not in GFP57 cells, two-color flow cytometry was applied with the use of a marker present only on the epithelial cell population. Cells were removed, resuspended, and reacted with antibody 323/A3; infected epithelial cells—defined as positive for both ESA and EGFP—were then enumerated. Results (Fig. 2Go, panels 9 and 10) show that 1.1% and 3.4% of cocultivated MCF7 and T47D cells, respectively, were ESA positive and EGFP positive, confirming infection by cell-to-cell contact. Control experiments, in which cocultivated breast cell monolayers with Daudi cells transiently expressed EGFP (Fig. 2Go, panels 11 and 12), yielded minimal background (<=0.10%) of dual-positive cells. The same level of background was seen when EGFP-expressing Daudi cells and T47D or MCF7 cells were mixed immediately before flow cytometry, suggesting that the background level did not arise during cocultivation (data not shown).

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 (0–40 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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