Superinfection of Epithelial Hybrid Cells (D98/HR-1, NPC-KT, and A2L/AH) withEpstein-Barr Virus and the Relationship to the C3d Receptor1
Toru Takimoto,2 Hiroshi Sato, Hisashi Ogura, Toshio Miyawaki, and Ronald (¡laser*
Department of Medical Microbiology and Immunology, The Ohio State University College of Medicine and Comprehensive Cancer Center, Columbus, Ohio 43210¡T.T., R. G.]; Department of Virology, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, 920 Kanazawa, Japan [H. S., H. O.f; Department ofPediatrics, School of Medicine, Kanazawa University, 13-1 Takaramachi, 920 Kanazawa, Japan [T. MJ; and Ohio State University Comprehensive Cancer Center,Columbus, Ohio 43210 [R. GJ
ABSTRACT
Three Epstein-Barr virus (EBV) genome-positive epithelial/hybrid celllines (D98/HR-1, NPC-KT, and A2L/AH) »eresuperinfected with EBVderived from P3HR-1 (HR-1), NPC-KT, and B95-8 cells. The HR-1
virus is lytic and induces early antigen in superinfected Raji cells; thevirus is not capable of transforming B-lymphocytes. Virus preparationsfrom NPC-KT cells have both transforming and early antigen-inducingproperties, while B95-8 virus can only transform B-lymphocytes. It waspossible to demonstrate EBV antigens after superinfection of D98/HR-1cells with both HR-1 virus and NPC-EBV. The NPC-KT hybrid cells,
which were originally prepared by fusing human adenoid epithelial cells(Ad-AH) and EBV genome-positive NPC explanted epithelial cells, weresusceptible to superinfection with HR-1 virus but not to NPC-EBV. TheA2L/AH hybrid cells, which were prepared by fusion between Ad-AHcells and lymphocytes transformed by B95-8 virus, could not be super-
infected with any of the virus preparations. In order to further investigatethe nature of the EBV receptor as it relates to other cell membranecomponents, we examined cell surface markers on Ad-AH, NPC-KT,A2L/AII, and D98/HR-1 cells using monoclonal antibodies and by rosetteformation. We found that the NPC-KT, A2L/AH, and Ad-AH cell lines
express the OKB2 antigen and that the common acute lymphoblasticleukemia antigen is expressed on the A2L/AH cells. We also found thatNPC-KT parental cells and a clone of NPC-KT cells express erythrocyte
antibody complement b and erythrocyte antibody complement d, asdetermined by rosette formation, but were negative for C3b and C3dwhen monoclonal antibodies against these two markers were used. TheD98/HR-1 cells were also confirmed to be negative for Oh and Od.
The data suggest that the Od receptor may be part of the EBV receptorbut that the C3d receptor, by itself, is not the only receptor to whichEBV can bind.
INTRODUCTIONThe EBV4 is a human oncogenic herpesvirus which has been
closely associated with two types of malignant disease, Burkitt's
lymphoma (1) and NPC (2). The EBV has primarily beenassociated with B-lymphocytes. It was thought that the in vitrohost range for EBV was strictly B-lymphocytes. However, several years ago we found that certain NPC epithelial expiant cellcultures and normal nasopharyngeal epithelial cells obtainedfrom squirrel monkeys could be directly infected with HR-1virus (3,4). Recently, Sixbey et al. (5) showed that some humanectocervical epithelial cells could be directly infected with "wildtype" EBV. The susceptibility of human epithelial cells to EBV
now shown in such studies strongly supports the hypothesisthat certain epithelial cells derived from the human nasophar-
Received 10/30/85; revised 1/27/86; accepted 1/29/86.The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by grant R810802 from the Environmental Protec
tion Agency and Grant CA 29066 from the National Cancer Institute.2On leave from the Department of Otorhinolaryngology, School of Medicine,
Kanazawa University, 13-1 Takaramachi, 920 Kanazawa, Japan.3To whom requests for reprints should be addressed.4The abbreviations used are: EBV, Epstein-Barr virus; EA, early antigen;
ynx should be susceptible to infection with EBV, though thisremains to be demonstrated.
Earlier studies have suggested that the EBV receptor wasclosely associated with the complement C3d receptor, a M,140,000 to 145,000 membrane glycoprotein (6). For example,it was demonstrated that EBV absorption by human cells couldbe blocked by pre-incubation of the EBV receptor positive cellswith C3, followed by rabbit anti-C3 and goat anti-rabbit IgGantibodies (7). In addition, several human cell lines, when testedfor C3 receptors and EBV receptors, either co-expressed orsimultaneously showed the absence of these receptors (7). However, in another study, it was shown that D98/HR-1 humanEBV genome positive epithelial hybrid cells could be superinfected with EBV derived from HR-1 cells (lytic nontransform-ing virus), and that these cells did not have detectable C3receptors (8). In this report, we confirm and extend this finding.
We succeeded in establishing an epithelial hypoxanthine-guanine-phosphoribosyltransferase-deficient cell line (Ad-AH)derived from the human nasopharynx (adenoid) (9), and weused this cell line to prepare epithelial/epithelial and epithelial/lymphoblastoid hybrid cells. Included in this library of hybridcell lines are two recently characterized human epithelial/na-sopharyngeal hybrid cell lines, NPC-KT and A2L/AH (9, 10),and the cell line originally examined for C3 receptors, thehuman epithelial/Burkitt's lymphoma hybrid cell line, D98/
HR-1 (11). The NPC-KT hybrid cells were obtained by fusingAd-AH cells with EBV genome-positive NPC tumor cells. Theyare a unique cell line because the cells produce biologicallyactive virus (NPC-EBV) that can transform lymphocytes andsuperinfect Raji cells (12, 13).
In this study, we have performed superinfection studies usingHR-l-EBV and virus obtained from NPC-KT cells and B95-8cells. We have shown previously that D98/HR-1 hybrid cellscan be superinfected with HR-l-EBV (8) but not with B95-8virus, and we now demonstrate that D98/HR-1 cells can alsobe superinfected with NPC-EBV. We found that the NPC-KThybrid cells can be superinfected with HR-l-EBV as well.Studies were performed to characterize several surface markerson Ad-AH, NPC-KT, A2L/AH, and D98/HR-1 hybrid cells inan attempt to study virus-cell interaction and to further explorethe relationship of the EBV and C3d receptors.
MATERIALS AND METHODS
Cells. The Ad-AH cells are an hypoxanthine-guanine-phosphoribo-syltransferase-deficient cell line derived from the human nasopharynx(adenoid) (9). The NPC-KT hybrid cell line (clone 1 and clone 2) wasderived from the fusion of Ad-AH cells and EBV genome-positive NPCepithelial expiant culture cells (10, 13). The A2L/AH hybrid cell linewas derived from the fusion of Ad-AH cells and human lymphocytestransformed in vitro by B95-8 virus (10). The D98/HR-1 hybrid cellline was derived from the fusion of D98 cells (a HeLa cell variant) andHR-1 cells (11). The epithelial/hybrid cell lines were maintained inEagle's medium containing 10% fetal bovine serum, gentamicin (25
i«g/ml),and 0.05% NaHCO,. The epithelial Ad-AH cells are EBV
genome negative, but all three epithelial hybrid cell lines are genomepositive, as demonstrated by the presence of Epstein-Barr virus nuclearantigen. D98/HR-1 cells do not spontaneously express EA or VGA,but the NPC-KT clone 1 and clone 2 and A2L/AH hybrid cellsspontaneously express EA/VCA at a low level (<0.05%). The B95-8transformed lymphoblastoid cell line (A2L) was maintained in RPMI1640 medium containing 10% fetal bovine serum, gentamicin (25 ng/ml), and 0.05% NaHCO3.
Virus. In order to study the interaction of EBV with differentepithelial/hybrid cells, in regard to infectivity. we used three biologicallydifferent isolates of EBV; HR-1, which is lytic and non-transforming(14); B95-8, which is a transforming strain (15); and NPC-EBV, whichcan either induce lytic replication in superinfected Raji cells similar toHR-1 virus, or transform human B-lymphocytes similar to B95-8 (13).
NPC-EBV was obtained from NPC-KT cells. Freshly trypsinizedNPC-KT clone 2 cells were seeded to approximately 80% confluencyin 75 cm2 plastic Falcon flasks and incubated in growth medium. After24 h at 35°C,the medium was changed, and growth medium containing
60 fjg 5-iododeoxyuridine/ml was added. The cultures were then incubated for 3 days at 35°C.The medium containing 5-iododeoxyuridine
was then removed, and the cells were washed with phosphate-bufferedsaline. Thereafter, the cells were incubated in normal growth mediumfor an additional 7 days at 35°C.The HR-1 and B95-8 cells were grown
in the growth medium containing ^-O-tetradecanoylphorbol-n-ace-tate (20 ng/ml) at 35°Cfor 7 days to increase virus synthesis. The
supcrnatants of NPC-KT clone 2, HR-1, and B95-8 cell cultures werecentrifuged to pellet the cells and then recentrifuged to pellet the virusand filtered through a 0.45-Mm filter, as described elsewhere (16). Viruswas resuspended in a volume of RPMI 1640 medium in order to achieve100 to 200x concentrations.
Supvrinfection of Cells with EBV. The epithelial/hybrid cells weregrown as monolayers on glass coverslips. Approximately 24 h afterculturing. EBV concentrates were added to each cell culture, andincubation was continued for l h at 37°C.After 3 ml of completeEagle's medium were added to the culture, the cells were incubated for4 days at 37"C and then fixed in acetone and examined for EA/VCA
(8).Immunofluorescence Techniques. Indirect IF was used to detect EBV-
spccific antigens in the superinfected epithelial/hybrid cells as describedpreviously (3, 8). Cells on coverslips were fixed with acetone andallowed to adsorb precharacterized EBV-positive human sera.
Assays of Cell Surface Markers. The epithelial/hybrid cells weredetached from the flasks by rubber policemen and dispersed by pipetting. The cells were analyzed for surface phenotype by indirect IF asdescribed elsewhere (17). In brief, 1 x 10' viable cells were treated with
100 /«Iof specific or control monoclonal antibodies at saturating concentrations, incubated at 4°Cfor 30 min, and washed 3 times. These
included OKB2 (Ortho Diagnostics Systems, Inc.), CALLA (CoulterElectronics, Inc.), C3b (Dakopatts, Denmark), and C3d (Coulter Electronics, Inc.). Cells were then treated with 100 ¿ilof a 1:40 dilution ofgoat anti-mouse IgG or goat anti-mouse IgM conjugated with fluores-cein ¡sothiocyanate and incubated at 4°Cfor 30 min. After washing,
each cell was analyzed for IF using a Coulter Epics clinical flowcytometer.
li and Complement Receptor Tests by EA and EAC. The rosette testswere performed as described elsewhere (7, 18). Fc receptors were scoredby rosette formation with IgG sensitized sheep RBC. C3b and C3dreceptors were detected by rosette formation with EAC cells sensitizedwith IgM.
RESULTS
Superinfection of D98/HR-1 Hybrid Cells with HR-1-,NPC-, and B95-8 EBVs. In order to study the nature of theEBV receptor and the expression of EBV antigens after super-infection, we attempted to superinfect three different EBVgenome positive epithelial hybrid cell lines with three differentbiological isolates of EBV.
The D98/HR-1 cells are positive for Epstein-Barr virus nu-
2542
clear antigen but do not spontaneously express EA/VCA (11).The cells were superinfected with HR-1 virus at a concentrationable to produce approximately 15% EA positive Raji cells 48 hpostinfection and examined for expression of EBV EA/VCAby using EA+/VCA* human sera in the indirect IF test 4 days
after superinfection. We found approximately 1-2% EA/VCA-positive cells (Table 1). We then attempted to superinfect theD98/HR-1 cells with NPC-EBV at a concentration able toproduce approximately 9% EA/VCA-positive Raji cells. Approximately 1% of D98/HR-1 cells superinfected with NPC-EBV were positive with EA+/VCA+ sera (Fig. 1). As reportedpreviously, we found that D98/HR-1 cells could not be super-infected with B95-8 virus (8).
Superinfection of NPC-KT Hybrid Cells with HR-1, NPC-, orB95-8 EBVs. Similar experiments were performed with NPC-KT cells that were superinfected with HR-1, NPC-, and B95-8viruses (Table 1). Less than 0.05% of mock-infected NPC-KTclone 1 and clone 2 cells were IF positive using Ea+/VCA*human sera. When the cells were superinfected with HR-1EBV, we found approximately 1% EA/VCA-positive cells (Fig.2). We did not find a significant increase in EA/VCA-positiveNPC-KT clone 1 and 2 cells when superinfected with NPC orB95-8 EBV.
Superinfection of A2L/AH Hybrid Cells with HR-1, NPC-KT,or B95-8 EBVs. Less than 0.05% of the A2L/AH cells spontaneously express EA/VCA. When A2L/AH cells were superinfected with HR-1, NPC-, and B95-8 EBVs, no increase in IF
Table 1 Percentage of cells expressing EA/ VCAfollowing EBV superinfectionThe frequency of EA/VCA-positive cells is shown 4 days after superinfection.
°Determined by the indirect IF test using monoclonal antibodies as describedin "Materials and Methods."
h KB cells were used as negative controls.' NT. not tested.
positive cells was observed, showing the lack of susceptibilityof these cells to all three isolates (Table 1).
Cell Surface Markers. In order to investigate the nature ofthe EBV receptor the NPC-KT and A2L/AH human naso-pharyngeal epithelial/hybrid cell lines were examined for theirreactivity with monoclonal antibodies to CALLA and OKB2.It is known that the OKB2 antigen is found on surface naso-pharyngeal epithelial cells (19). All epithelial/nasopharyngealcells were reactive with OK.B2 monoclonal antibody (Table 2).CALLA (a pre-B-cell marker) is expressed on approximately38% of A2L/AH hybrid cells, whereas Ad-AH cells, NPC-KTclone 1 and clone 2, or KB cells used as control did not reactwith this monoclonal antibody as expected.
Using the rosette test, the Fc receptor was not expressed onthe surface of NPC-KT, A2L/AH, and Ad-AH cell lines. Wefound that EACb and EACd receptors were detected by resetting on NPC-KT clone 1 cells and the controls but not on NPC-KT clone 2 cells, A2L/AH, and Ad-AH cells. However, wewere not able to demonstrate C3b or C3d receptors usingspecific monoclonal antibodies to both markers on NPC-KT,clone 1 or clone 2, A2L/AH, or D98/HR-1 cells; D98 cells, thehuman epithelial (a HeLa cell variant) parent of D98/HR-1cells, were also negative, as expected. Using the same reagents,the control Raji cells were >90% C3d positive, and B95-8transformed cord blood lymphocytes were >90% C3b positive.
DISCUSSION
We previously established an epithelial/BL hybrid cell line(D98/HR-1) derived from the fusion of D98 human epithelialcells and HR-1 cells, and we have used these cells to study theexpression and regulation of the EBV genome in epithelial cells
(11). We showed that the D98/HR-1 cells could be directlysuperinfected with HR-l-EBV, although they lacked detectable
C3 receptors, as measured by rosetting (8). These results led tostudies where we succeeded in directly superinfecting explantedNPC epithelial cells and normal squirrel monkey nasopharyn-geal epithelial cells (3,4). It followed from these data that someepithelial cells, especially nasopharyngeal epithelial cells, mostlikely have the receptor for EBV. We have continued to studyepithelial/hybrid cells and recently succeeded in establishingtwo EBV-genome-positive epithelial/nasopharyngeal hybridcell lines (9, 10).
These cells have been used to explore the nature of the EBVreceptor. We have shown in this report that the NPC hybridcell line (NPC-KT) can be directly superinfected at a low levelwith HR-l-EBV but not NPC-EBV and that the D98/HR-1hybrid cells can be superinfected with NPC-EBV as well as HR-l-EBV. The genetic information to code for the EBV receptoron the surface of NPC-KT cells was contributed by the NPCepithelial parent cells, since the other parent cells, Ad-AH, arenot infectable of EBV. These data further support the possibilitythat certain nasopharyngeal epithelial cells possess the properreceptor for EBV. While the percentage of IF positive cells inthese experiments is low, the fact that the cells are infectablemay provide a clue to the interaction of EBV with epithelialcells.
It is possible that the reason for some of the past difficultyin showing routine infection of human epithelial cells may notonly be related to the host range for EBV but also may bedependent on the strain of EBV used (5). Data from severalstudies have shown that certain epithelial cells can support thereplication of EBV after direct infection (20). It is possible, asSixbey et al. (5) pointed out recently, that a modification(s) invirus obtained from laboratory cell lines might have occurred,thereby selecting for a specific kind of virus in regard to infec-tivity and host range. Until now, all laboratory strains werederived from lymphoblastoid cells, and it is therefore possiblethat the structure of the envelope of laboratory strains and/ormodifications of the virus genome may be different from thatof virus released from oropharyngeal epithelial cells. Thesedifferences could be related, at least in part, to infectivity.
In this study, we found that the NPC-EBV was not capableof superinfecting NPC-KT clone 1 and clone 2 cells, but NPC-KT cells could be superinfected with HR-1 EBV. Both HR-1and NPC-EBV can superinfect D98/HR-1 cells. It is possiblethat the NPC-EBV genome in NPC-KT cells may inhibit thesuperinfection by NPC-EBV. The mechanism of EA inductionafter superinfection is at present unclear. Some investigatorssuggest that EA induction after superinfection may be relatedto complementation or recombination of superinfecting virusDNA with the resident virus genome (21, 22). Other studiessuggest that recombination does not take place and that heter-ogenous virus DNA (in the HR-1 virus preparations) is what isresponsible for virus expression after superinfection (23). Further studies will need to be performed to clarify these discrepancies. If there are at least two types of virus DNA in preparations of HR-1 EBV, as suggested (24), then it is possible that:(a) D98/HR-1 cells can be superinfected and EA be expressedbecause of heterogeneity between exogenous and endogenousHR-1 virus DNA, which may not be the case for NPC-KT Cellssuperinfected with NPC virus; or (b) NPC-KT cells may possessthe receptor for HR-1 virus alone. Studies on the heterogeneityof NPC virus DNA and the EBV receptor on NPC-KT cellsare under way.
Previously, we reported a specific marker, Rbt(2q;6p), on2543
chromosomes of Ad-AH, A2L/AH, and NPC-KT cells, whichnone of the other cell lines in our laboratory have (25), and wehave used this marker to rule out contamination of our celllines with other lymphoblastoid cell lines.
In this study, we have examined cell surface markers on thesecell lines and examined the association of the EBV receptorand its link to the C3d receptor. We found that all nasophar-yngeal epithelial cells studied are reactive with the OKB2 monoclonal antibody. This antibody is expressed on human tonsilepithelium as well as B-lymphocytes (19). The epithelial Ad-AH cells are derived from nasopharyngeal tonsil (adenoid). TheNPC-KT Cl 1 cells reacted with EACb and EACd, whereasmonoclonal antibodies against C3b and C3d did not detectthese markers. It is possible that only a portion (those responsible for rosetting) of the determinant of the C3d receptor isexpressed on the surface of NPC-KT clone 1 cells. Interestingly,the percentage of EA/VCA synthesis after superinfection ofEAC-positive NPC-KT clone 1 cells with HR-l-EBV was essentially the same as that after superinfection with EAC-nega-tive NPC-KT clone 2 cells.
In a previous study, we showed that D98/HR-1 did notexpress the C3 receptor using a rosetting procedure and thatthe cell line could be superinfected with HR-1 virus (8). Wehave now shown that they are negative for C3b and C3d usingmonoclonal antibodies. We suggested the possibility that: (a)the C3 receptor is present on the cell membrane but cannot bedetected because of a "masking substance;" or (b) the expression
of the C3 receptor is blocked at the level of synthesis, reflectingsome interaction between the two genomes present in the hybridnucleus, and/or at insertion in cell membranes. In this studywe have found that A2L/AH hybrid cells did not express theC3 receptor, whereas A2L lymphoblastoid cells did express theC3 receptors (data not shown). The NPC-KT clone 1 cellsexpress a partial C3 receptor, while the NPC-KT clone 2 cellsare negative. We are left with the conclusion that the C3dreceptor may be part of the EBV receptor but that it, by itself,is not the only receptor that EBV can bind to on the cell surface.By using epithelial/hybrid cells similar to the three cell linesdescribed in this study, it may be possible to learn more aboutthe EBV receptor and the association of EBV to NPC.
ACKNOWLEDGMENTS
We thank Dr. S. Natsuume-Sakai of the Department of Immuno-biology. Cancer Research Institute, Kanazawa University, Kanazawa,Japan, for providing us with C3b and C3d indicator cells.
REFERENCES
1. Epstein. M. A.. Achong. B. G.. and Barr. Y. M. Virus particles in culturedlymphoblasts from Kmkin'-, lymphoma. Lancet. /: 702-703, 1964.
2. Henle. W.. Ho. J. H. C.. Henle. G.. and Kwan, H. C. Antibodies to Epstein-ll.iii virus related antigens in nasopharyngeal carcinoma. Comparison ofactive cases and long term survival. J. Nati. Cancer Inst., 51: 1398-1412,1973.
4. Glaser, R.. Lang, C. M., Lee. K. J., Schuller, D. E., Jacobs, D., andMcQuattie. C. Attempt to infect nonmalignant nasopharyngeal epithelialcells from humans and squirrel monkeys with Epstein-Barr virus. J. Nati.Cancer Inst., 64: 1085-1090, 1980.
5. Sixbey. J. W., Vesterinen, E. H., Nedrud, J. G., Raab-Traub, N., Walton. L.A., and Pagano. J. S. Epstein-Barr virus replication in human epithelial cellsinfected in vitro. Nature (Lond.), 306:480-483, 1983.
6. Frade. R.. Barel. M., Ehlin-Henriksson, B., and Klein, G. gpl40, the C3dreceptor of human B lymphocytes, is also the Epstein-Barr virus receptor.Proc. Nati. Acad. Sci. USA, «2:1490-1493, 1985.
7. Jondal. M., Klein. G., Oldstone, M., Bokish, V., and Yefenof, E. Surfacemarkers on human B- and T-lymphocytes. VIII. Association between complement and Epstein-Barr virus (EBV) receptors on human lymphoid cells.Scand. J. Immunol.. 5:401-410. 1976.
9. Takimoto, T., Furukawa, M., Hatano, M., and Umeda, R. Epstein-Barr virusnuclear antigen-positive nasopharyngeal hybrid cells. Ann. Otol. Rhinol.Laryngol., 939: 166-169. 1984.
10. Takimoto, T., Kamide. M., and Umeda, R. Establishment of Epstein-Barrvirus (EBV)-associated nuclear antigen (EBNA)-positive nasopharyngeal carcinoma hybrid cell line (NPC-KT). Arch. Otorhinolaryngol.. 239: 87-92,1984.
11. Glaser, R., and Rapp, F. Rescue of Epstein-Barr virus from somatic cellhybrids of Burkitt lymphoblastoid cells. J. Virol., 10: 288-296, 1972.
12. Takimoto, T., Ogura, H., Sato, H., and Hatano. M. A new strain of Epstein-Barr virus derived from nasopharyngeal carcinoma hybrid cells. Gann. 75:947-949. 1984.
13. Takimoto. T., Ogura. H., Sato. H., Umeda. R., and Hatano, M. Isolation oftransforming and early antigen-inducing Epstein-Barr virus from nasophar-yngeal carcinoma hybrid cells (NPC-KT). J. Nati. Cancer Inst., 74: 57-59,1985.
14. I limitila. Y., Kohn, M., Yamaguchi, J., Wudarski. D. J., Blakeslee, J., Jr.,and Grace, J. T., Jr. Immunofluorescence and herpes-type virus particles inthe P3HR-1 Burkitt lymphoma cell line. J. Virol., /: 1045-1051, 1967.
15. Miller, G., Robinson, J., Heston, L., and Lipman. M. Difference betweenlaboratory strains of Epstein-Barr virus based on immortalization, abortiveinfection and interference. Proc. Nati. Acad. Sci. USA, 71:4006-4010. 1974.
16. Traul, K. A., Stephens, R., Gerber, P.. and Peterson. D. Productive Epstein-Barr viral infection of the human lymphoblastoid cell line 1640 with releaseof early antigen inducing and transforming virus. Int. J. Cancer, 20: 247-255, 1977.
17. Anderson, K. C.. Bates, M. P., Slaughenhoupt. B. L.. Pinkus, G. S., Schloss-man. S. F.. and Nalder, L. M. Expression of human B-cell-associated antigenson leukemias and lymphomas: a model of human B-cell differentiation.Blood. 63: 1424-1433, 1984.
18. Jondal, M. Surface markers on human B- and T-lymphocytes. IV. Distribution of surface markers on resting and blast-transformed lymphocytes. Scand.J. Immunol., 3: 739-747. 1974.
19. Knowles. D. M.. II. Tolidjian. B., Marboe. C. C.. Mittler. R. S.. Talle, M.A., and Goldstein. G. Distribution of antigens defined by OKI! monoclonalantibodies on benign and malignant cells and on nonlymphoid tissues. Blood.63:886-896. 1984.
20. Glaser. R., Noyes, !.. and Milo, G. Pathogenesis of Epstein-Barr virusinfection: the host-range of EBV now includes epithelial cells. In: F. Rapp(ed.), Herpesvirus. New York: Alan R. Liss, Inc., 1984.
21. Fresen, K. O., C o, M. S.. Gissman, L.. and zur Hausen, H. NC37-R1 EB-virus: a possible ecombinant between intracellular NC37 viral DNA andsuperinfectingP3HR-l EBV. Intemrology, 12: 303-310. 1979.
22. Stoerker, J.. Yajim,1 N'., and Glaser. R. The interaction of non-transforming
Epstein-Barr virus (EBV) with cell-associated EBV DNA in superinfectedlymphoblastoid cell lines. Int. J. Cancer., 30: 385-392. 1982.
23. Miller, G., Rabson. M., and Heston, L. Epstein-Barr virus with heterogeneous DNA disrupts latency. J. Virol., 50: 174-182, 1984.
24. zur Hausen, H., and Fresen, K. O. Heterogeneity of Epstein-Barr virus. II.Induction of early antigens (EA) by complementation. Virology. 81: 138-143, 1977.
25. Takimoto. T.. Ogura. M Ohno, S.. Umeda, R.. and Hatano. M. Tumori-genicity of nasophary >•I carcinoma hybrid cell line. J. Nati. Cancer Inst.,73:711-715, 1984.
![Page 1: Superinfection of Epithelial Hybrid Cells (D98/HR-1, NPC-KT ......[CANCER RESEARCH 46, 2541-2544, May 1986] Superinfection of Epithelial Hybrid Cells (D98/HR-1, NPC-KT, and A2L/AH)](https://reader035.documents.pub/reader035/viewer/2022071109/5fe3bdc1fcad73513005cb3f/html5/thumbnails/1.jpg)
![Page 2: Superinfection of Epithelial Hybrid Cells (D98/HR-1, NPC-KT ......[CANCER RESEARCH 46, 2541-2544, May 1986] Superinfection of Epithelial Hybrid Cells (D98/HR-1, NPC-KT, and A2L/AH)](https://reader035.documents.pub/reader035/viewer/2022071109/5fe3bdc1fcad73513005cb3f/html5/thumbnails/2.jpg)
![Page 3: Superinfection of Epithelial Hybrid Cells (D98/HR-1, NPC-KT ......[CANCER RESEARCH 46, 2541-2544, May 1986] Superinfection of Epithelial Hybrid Cells (D98/HR-1, NPC-KT, and A2L/AH)](https://reader035.documents.pub/reader035/viewer/2022071109/5fe3bdc1fcad73513005cb3f/html5/thumbnails/3.jpg)
![Page 4: Superinfection of Epithelial Hybrid Cells (D98/HR-1, NPC-KT ......[CANCER RESEARCH 46, 2541-2544, May 1986] Superinfection of Epithelial Hybrid Cells (D98/HR-1, NPC-KT, and A2L/AH)](https://reader035.documents.pub/reader035/viewer/2022071109/5fe3bdc1fcad73513005cb3f/html5/thumbnails/4.jpg)
![Page 5: Superinfection of Epithelial Hybrid Cells (D98/HR-1, NPC-KT ......[CANCER RESEARCH 46, 2541-2544, May 1986] Superinfection of Epithelial Hybrid Cells (D98/HR-1, NPC-KT, and A2L/AH)](https://reader035.documents.pub/reader035/viewer/2022071109/5fe3bdc1fcad73513005cb3f/html5/thumbnails/5.jpg)
![37. Holding Company [D98-J14] [27.02.2014]](https://static.documents.pub/doc/80x56/5695cf0a1a28ab9b028c5394/37-holding-company-d98-j14-27022014.jpg)
