Proc. Nat. Acad. Scd. USA Vol. 73, No. 3, pp. 960-963, March 1976 Microbiology Superinfection of epithelial nasopharyngeal carcinoma cells with Epstein-Barr virus (induction/epithelial cells/virus receptors) RONALD GLASER*, GUY DE THOt, GILBERT LENOIRt, AND JOHN H. C. Hot * Department of Microbiology, The Milton S. Hershey Medical Center, The Pennsylvania State University, College of Medicine, Hershey, Pa. 17033; t International Agency for Research on Cancer (IARC), 150 Cours Albert Thomas, 69008 Lyon, France; and t Medical and Health Department, Institute of Radiology, Queen Elizabeth Hospital, Wyley Road, Kowloon, Hong Kong Communicated by Werner Henle, December 11, 1975

ABSTRACT Attempts were made to superinfect epitheli- that tumor could be superinfected with EBV and that the al explant cell cultures prepared from nasopharyngeal carci- resident EBV genome could be induced with IdUrd (17). nomas with Epstein-Barr virus. Virus-specific markers were reported here was performed as an extension of observed in such cultures 3 days after superinfection. In ad- The study dition, expression of Epstein-Barr virus early antigens was these preliminary results and previous work with Burkitt/ observed in epithelial cell explant cultures treated with io- epithelial hybrid cells to determine how EBV infects epithe- dodeoxyuridine. The results suggest that epithelial cells of at lial cells of the nasopharynx and to clarify the role of EBV in least certain nasopharyngeal carcinomas possess the receptor NPC. for the Epstein-Barr virus and that the latent virus genome can be induced from epithelial cells prepared from such tu- mors. MATERIALS AND METHODS Preparation of NPC Epithelial Cell Explants. Tumor Data obtained from a variety of studies support an associa- specimens classified as typical NPC from patients in Hong tion between the Epstein-Barr virus (EBV) and nasopharyn- Kong (patient numbers 75-1525, 75-1531-1, 75-1531-2, and geal carcinoma (NPC). Results of immunofluorescence (IF) 75-1576) were cut into approximately 1 mm pieces. The tests have shown that patients with NPC usually possess high tumor specimens were placed on glass coverslips in 13 mm titers of antibody directed against EBV-specific virus capsid plastic tissue culture plates (Falcon) which had been pre- antigens (VCA) (1), membrane antigen (2), early antigen(s) treated with calf serum to aid in attachment of explants. A (3), soluble antigen(s) (4), and nuclear antigen (5). In addi- small volume of RPMI 1640 medium supplemented with tion, EBV-specific antibody levels have been shown to be re- 20% fetal calf serum, 100 units of penicillin per ml, and 100 lated to the clinical stage of the tumor (1, 5). ,ug of streptomycin per ml was added to the tissue culture When NPC tumor biopsy specimens were assayed for plates. Epithelial cells from the explants were allowed to EBV DNA by nucleic acid hybridization, virus-specific grow out on glass coverslips prior to treatment or superinfec- DNA was found in the specimens (6). Moreover, it has now tion. been shown that both EBV genomes (7) and the EBV-associ- Superinfection of NPC Epithelial Cells with EBV. ated nuclear antigen (EBNA) (8) are associated with the epi- Seven days after preparation of the NPC explants, the medi- thelial elements of NPC (9, 10) rather than with the infiltrat- um was changed and cells were grown in fresh RPMI 1640 ing lymphoid cells (11). medium, as previously described, for 24 hr. Coverslip ex- We have studied the expression and regulation of EBV in plant cell cultures were infected with 0.2 ml aliquots con- mammalian cells. To circumvent the inability of EBV to ly- taining 105 fluorescence-forming units (18) of EBV derived tically infect (or transform) cells other than B (bone-mar- from HR-1 cells. Virus was allowed to adsorb to the cells for row-derived) lymphocytes (12), we made somatic cell hy- 1 hr at 370, and 2 ml of complete RPMI 1640 medium was brids of Burkitt tumor cells (13) in order to get the EBV ge- added. The cells were incubated for 3 days at 370 and fixed nome into other cell types. The EBV genome could be main- in acetone for 10 min in preparation for immunofluores- tained in a repressed state in epithelial hybrid cells for long cence. periods of time and, under certain conditions, could be ex- Treatment of NPC Cells with IdUrd. To determine pressed in these cells (14, 15). Recent data suggest that EBV whether the repressed EBV genome in epithelial NPC cells expression in epithelial hybrid cells following induction with could be induced to express EBV early antigen (EA), studies iododeoxyuridine (IdUrd) is enhanced over that found in similar to those previously performed with Burkitt hybrid producer lymphoblastoid cell lines (HR-1) (16), and that this cells (14, 15) were conducted with IdUrd. NPC epithelial ex- observation may resemble a Ciost range phenomenon typical plants were prepared as described in the superinfection of other viruses. study. Twenty-four hours after initial change of medium, Since data suggest that EBV is a B lymphocyte tropic complete Eagle's medium containing 60 ,tg/ml of IdUrd virus, the way in which EBV enters and transforms epitheli- was added to the cultures. The cells were incubated for 3 al cells of the nasopharynx to induce NPC is a paradox. Pre- days at 370 and were fixed in acetone for 10 min; they were liminary data obtained from a study with one NPC tumor later examined for EBV-specific markers by the direct IF specimen suggest that epithelial cell explants prepared from test. Immunofluorescence Test. The direct IF test was used to detect EBV-specific EA in order to avoid nonspecific fluo- Abbreviations: EBV, Epstein-Barr virus; NPC, nasopharyngeal car- tumor ex- cinoma; EA, early antigen; VCA, virus capsid antigen; EBNA, Ep- rescence due to immunoglobulin G (IgG) in the stein-Barr-associated nuclear antigen; IF, immunofluorescence; B plants. Previously characterized EBV-positive serum from a lymphocyte, bone-marrow-derived lymphocyte. Tunisian NPC patient (TU 201) was conjugated directly to 960 Downloaded by guest on September 26, 2021 Microbiology: Glaser et al. Proc. Nat. Acad. Sci. USA 73 (1976) 961 RESULTS Superinfection of NPC epithelial cells with EBV Recent data suggest that epithelial/Burkitt hybrid cells, D98/HR-1, not only possess the receptor for EBV attach- ment, but are also superinfectable with EBV as demon- strated by the detection of EBV EA in a small percentage of cells (Glaser et al., unpublished data). As a result of this finding, attempts were made to superinfect epithelial cells prepared from NPC tumor specimens. Epithelial explants prepared from four NPC tumor biopsies obtained from three patients were grown on glass coverslips as described; one tumor (75-1531-2) was a second biopsy specimen taken approximately 4 weeks after the initial biopsy. Each cover- slip contained two separate explants from which cells had grown out. A preparation of 105 fluorescence-forming.units of EBV derived from the producer HR-1 cell line was added FIG. 1. Immunofluorescent photomicrograph of an epithelial to cell cultures which were then incubated for 3 days at 370. cell explant (tumor 75-1531-1) superinfected with EBV derived After fixation with acetone, the cells were examined for from HR-1 cells. Note EBV-specific EA in several cells. Approxi- EBV EA using the direct IF test. Three of four explants pre- mately X960. pared from tumor 75-1531-1 and infected with EBV demon- fluorescein isothiocyanate (FITC) and adsorbed to explant strated an average of 25% positive cells (Fig. 1 and Table 1). cell cultures. The cells were washed, mounted in glycerol on Most EA-positive cells showed nuclear fluorescence, though glass slides, and examined with a Leitz microscope with an whole cell fluorescence was also observed. Tumors 75-1576 ultraviolet light source. and 75-1525 were negative. Similar results were obtained on Characterization of Serum TU 201. Since a direct conju- epithelial explant cultures prepared from the second biopsy gate of serum TU 201 was used in the IF test, and since NPC specimen, 75-1531-2, taken approximately 1 month later sera often possess nonspecific anti-nuclear antibodies (19), (Table 1). Explant cell cultures not superinfected with EBV the serum was tested for such activity. The following cells but tested with the same direct conjugate serum were rega- were used to test serum TU 201: MOLT (an EBV-negative tive for EBV EA (Fig. 2). lymphoblastoid line), HeLa, human embryonic lung (HEL) cells, human embryonic nasopharyngeal (HEN) cells, fibro- Antigen induction in NPC epithelial explants blast cells derived from explants from two NPC tumors (Tu- Epithelial explants prepared from the same NPC tumor nisian TU 66 and TU 54), touch preparations of tumor 75- biopsies were grown out on glass coverslips and exposed to 1531-1 (the tumor in which EBV EA had been observed 60 gg/ml of IdUrd. After previous failure to recover EBV after superinfection and induction), and D98 cells. antigens from other tumor preparations, explants from

FIG. 2. Immunofluorescence photomicrograph of a mock-infected epithelial cell explant (tumor 75-1531-2). Note absence of EBV-posi- tive cells. Approximately X2200. Downloaded by guest on September 26, 2021 962 Microbiology: Glaser et al. Proc. Nat. Acad. Sci. USA 73 (1976) Table 1. Expression of EBV markers in epithelial cell Table 2. Assay for nonspecific anti-nuclear and explant cultures superinfected with EBV or anti-cytoplasmic antibodies in serum TU 201 treated with IdUrd IF test used % EBV-positive cells Cell type ACIF* Indirectt Directt Biopsy after treatment* % cellsEBNA-positivein touch (explants) IdUrdt EBVf preparations MOLT - - - HeLa - - 75-1531-1 5-10 25 >90 HEL - - - 75-1531-2 10-15 10-15 70-80 HEN - - - 75-1576 - 20-30 TU 66 - - - 75-1525 - 20-30 TU 54 - - - 75-1531-1§ NT NT - * These numbers represent the average of EBV-positive cells from D98 - cell explants examined. t 60 ,g/ml in Eagle's medium for 3 days at 370. ACIF, anti-complement immunofluorescence; NT, not tested. t 105 fluorescence-forming units of EBV derived from HR-1 cells. * Serum diluted 1:8. t Serum diluted 1:5. which only a small number of cells had grown out were t Direct conjugate diluted 1:5. used. It was thought that these cells would be the most ac- § Touch preparations of biopsy. tively dividing and that this might be important for incorpo- ration of IdUrd into cellular DNA. EBV-specific EA was ob- plasmic antibodies. Results are shown in Table 2. No nonspe- served primarily in the nucleus of 5-10% of the epithelial cific fluorescence was observed when serum TU 201 was NPC cells in tumor specimen 75-1531-1, and in 10-15% of tested on the aforementioned cells by the indirect IF test, the positive cells in tumor specimen 75-1531-2, after a 3-day the anti-complement immunofluorescence test, or the direct exposure to IdUrd (Fig. 3 and Table 1). Cells showing cyto- test. Touch preparations assayed from tumor 75-1531-1 were plasmic (whole cell)- fluorescence were also observed in ap- negative, as were untreated explant cultures prepared from proximately 1% of the total positive cells. The pattern of flu- tumor 75-1531-2 using serum TU 201 (Fig. 2). Therefore, orescence observed in the NPC epithelial cells was similar to this serum did not contain nonspecific anti-nuclear or anti- that observed in D98/HR-1 epithelial/Burkitt hybrid cells in cytoplasmic antibodies against a variety of human cell which EBV EA was expressed after treatment with IdUrd types or against untreated NPC explant cultures detectable (15). As in the superinfection study, explant cell cultures of by the IF test. tumor 75-1531-2 not treated with IdUrd but tested with the same direct conjugate serum were negative. Presence of EBNA in epithelial NPC explants The tumors were examined for EBNA activity using touch Characterization of serum TU 201 preparations prepared from each biopsy according to pre- The direct conjugate serum (EA-, VCA-positive) was tested viously published procedure (8-10). Results are given in for the presence of nonspecific anti-nuclear and anti-cyto- Table 1. Tumors 75-1525 and 75-1576, which had not re-

FIG. 3. Immunofluorescence photomicrograph of an epithelial cell explant (tumor 75-1531-1) treated with IdUrd for 3 days. Note EBV EA-positive cells. Approximately X2200. Downloaded by guest on September 26, 2021 Microbiology: Glaser et al. Proc. Nat. Acad. Sci. USA 73 (1976) 963 sponded to treatment with IdUrd or superinfection oonj thank Fred Rapp for constructive criticism of the manuscript and tained 20-30% EBNA-positive cells. Tumor 75-1531-1 con- Tamar Stematsky for discussion related to the study. This study was tained >90% EBNA-positive cells and tumor 75-1531-2 con- conducted under Contract N01 CP 43296 and Contract N01 CP tained 70-80% EBNA-positive cells. 53516 within the Virus Cancer Program of the National Cancer In- stitute, U.S. Public Health Service and by Public Health Service Re- DISCUSSION search Grant CA 15038-02 from the National Cancer Institute. R. Glaser is the recipient of a Leukemia Society Scholar award. This Since it has not been possible to infect non-lymphoblastoid study was performed at the IARC, Lyon, France, under the spon- cell types with EBV and since data concerning the host sorship of the Fogarty International Center, the National Institutes range for EBV suggest that it is a B lymphocyte tropic virus of Health, and the Institut National de la Sante et de la Recherche (9), the way in which EBV enters and transforms epithelial Medicale, Paris, France. cells of the nasopharynx is unknown. From studies with Burkitt hybrid cells we know that, once EBV gets into an ep- ithelial-like cell, it can be maintained in a repressed state for 1. Henle, W., Henle, G., Ho, H. C., Burtin, P., Cachin, Y., Clif- years and can be induced to replicate after treatment with ford, P., de Schryver, A., de The, G., Diehl, V. & Klein, G. IdUrd (14-16). Can in vivo cell fusion take place in the na- (1970) J. Nat. Cancer Inst. 44, 225-231. sopharynx, i.e., can the EBV genome in B lymphocytes be 2. de Schryver, A., Friberg, S., Jr., Klein, G., Henle, W., Henle, transferred into epithelial cells with subsequent transforma- G., de The, G., Clifford, P. & Ho, H. C. (1969) Clin. Exp. Im- tion It munol. 5, 443-459. (20)? has been shown by electron microscopy that 3. Henle, W., Ho, H. C. & Kwan, H. C. (1973) J. Nat. Cancer there may be loss of cell membrane integrity between epi- Inst. 51, 361-369. thelial and lymphoid cells in normal mucosa of the naso- 4. de-The, G., Sohier, R., Ho, H. C. & Freund, R. (1973) Int. J. pharynx and in NPC tumors (21, 22). Whether transfer of Cancer 12,368-377. EBV genetic material occurs under these circumstances re- 5. de-The, G., Ho, H. C., Ablashi, D. V., Day, N. E., Macario, A. mains to be established. J. L., Martin-Berthelon, M.Cl. & Sohier, R. (1975) Int. J. Can- We have data that suggest that a small percentage of epi- cer 16, 713-721. thelial D98/HR-1 Burkitt hybrid cells possess the receptor 6. zur Hausen, H., Schulte-Holthausen, H., Klein, G., Henle, W., for EBV Henle, G., Clifford, P. & Santesson, L. (1970) Nature 228, (Glaser et al., unpublished data). These data sup- 1056-1058. port the concept that if epithelial cells have the proper re- 7. Wolf, H., zur Hausen, H. & Becker, V. (1973) Nature New ceptor for EBV, then infection with EBV is possible. Data Biol. 244,245-247. presented in this report extend these findings and reinforce 8. Reedman, B. M. & Klein, G. (1973) Int. J. Cancer 11, 499- the notion that epithelial cells of the nasopharynx (progeni- 520. tors of the carcinoma cells), in at least certain individuals, 9. Huang, D. P., Ho, J. H. C., Henle, W. & Henle, G. (1974) Int. possess the proper receptor for EBV. We have shown that J. Cancer 14,580-588. EBV EA can be synthesized by the resident EBV genome in 10. Desgranges, C., Wolf, H., de The, G., Shanmugaratnam, K., NPC tumor cells treated with IdUrd. The cells in which EA Cammoun, N., Ellouz, R., Klein, G., Lennert, K., Mufioz, N. & was observed were epithelial in morphology, confirming by zur Hausen, H. (1975) Int. J. Cancer 16,7-15. another procedure (induction using IdUrd) the association of 11. de-The, G., Ablashi, D. V., Liabeuf, A. & Mourali, N. (1973) EBV Biomedicine 19,349-352. the genome and the epithelial elements of NPC tu- 12. Pattengale, P. K., Smith, R. W. & Gerber, P. (1974) J. Nat. mors. It is not clear whether VCA is synthesized in IdUrd- Cancer Inst. 52, 1081-1086. treated or superinfected NPC cells, since the only direct 13. Glaser, R. & O'Neill, F. J. (1972) Science 176, 1245-1247. conjugate serum available was EA- and VCA-positive. Fur- 14. Glaser, R. & Rapp, F. (1972) J. Virol. 10, 288-296. ther studies to clarify this point can be performed using a 15. Glaser, R., Nonoyama, M., Decker, B. & Rapp, F. (1973) Viro- VCA-specific direct conjugate serum. The fact that EBV has logy 55, 62-69. been shown to be "shed" in the oropharynx (23) places the 16. Glaser, R., Farrugia, R. & Brown, N. (1975) Virology, in press. virus in the vicinity of nasopharyngeal cells, and infection of 17. Glaser, R., Lenoir, G., Ho, J. H. -C. & de The, G. (1975) these cells may be possible. Biomed. Express, in press. The two NPC biopsies taken in which EBV EA was induc- 18. Mele, J., Glaser, R., Nonoyama, M., Zimmerman, J. & Rapp, ible with IdUrd F. (1974) Virology 62, 102-111. and superinfectable with EBV may have 19. Yoshida, T. 0. (1971) in Recent Advances in Human Tumor been unusual; more tumor biopsies will have to be studied. Virology and Immunology, eds. Nakahara, W., Nishioka, K., The high percentage of EBNA-positive cells may have been Hirayama, T. & Ito, Y. (University of Tokyo Press, Tokyo), a determining factor in the receptivity of these cells to the pp. 443-460. two treatments; it is also possible that this may have en- 20. Henle, W. & Henle, G. (1973) Cancer Res. 33, 1419-1423. hanced detectability of virus expression, since the two non- 21. Gazzolo, L., de The, G., Vuillaume, M. & Ho, H. C. (1972) J. infectable and noninducible tumors contained <30% EBNA- Nat. Cancer Inst. 48, 73-86. positive cells. 22. Vuillaume, M. & de The, G. (1973) J. Nat. Cancer Inst. 51, 67-80. The authors acknowledge the excellent technical assistance of M. 23. Miller, G., Niederman, J. C. & Andrews, L. (1973) N. Engl. J. F. Lavoue, M. C. Favre, C. Piccoli, and G. Baughman. The authors Med. 288,229-232. Downloaded by guest on September 26, 2021