Proc. Natl. Acad. Sci. USAVol. 86, pp. 9524-9528, December 1989Medical Sciences

Two epithelial tumor cell lines (HNE-1 and HONE-1) latentlyinfected with Epstein-Barr virus that were derived fromnasopharyngeal carcinomasRONALD GLASER*tt, HAI-YING ZHANG*, KAITAI YAO§, HE-CHENG ZHU§, Fu-Xi WANG§, GUI-YUAN LI§,DONG-SENG WEN§, AND YING-PING LI§*Department of Medical Microbiology and Immunology, The Ohio State University Medical Center, Columbus, OH 43210; tComprehensive Cancer Center,The Ohio State University, Columbus, OH 43210; and %Cancer Research Laboratory, Hunan Medical University, Changsha, Hunan, People'sRepublic of China

Communicated by Leo A. Paquette, August 16, 1989 (received for review April 26, 1989)

ABSTRACT Two epithelial tumor cell lines were estab-lished from biopsy specimens of nasopharyngeal carcinomas(NPC). The specimens were taken from poorly differentiatedsquamous cell carcinomas ofthe nasopharynx. The tissues wereprepared for cell culture and eventually two continuous epi-thelial cell lines were obtained and designated HONE-1 andHNE-1. Light and electron microscopic examination of thesetwo cell lines demonstrated cells with an epithelial morphologyincluding the presence of desmosomes. The HNE-1 cell line hasbeen passaged more than 100 times and the HONE-1 cell linehas been passaged more than 90 times. It was found thatearly-passage uncloned HNE-1 cells (passage 23) could besuperinfected with the B95-8 and NPC-EBV isolates as dem-onstrated by the induction of Epstein-Barr virus (EBV)-specific early antigen(s) in a small percentage of the cells;HONE-1 cells could also be superinfected with EBV. Southernblot analysis detected EBV DNA in samples from unclonedHNE-1 cells at passages 12, 17, 21, 27, and 35. However, bypassage 45, EBV DNA could no longer be detected in HNE-1cells by Southern blot analysis. The EBV genome was detectedin parental HONE-1 cells at subculture 9 and in clone 40 cellsup to passage 40 thus far. When HNE-1 cells were examined forthe expression of the EBV-encoded nuclear antigen (EBNA) atpassage 12, only about 10% of the cells were found to bepositive. The percentage of EBNA-positive HNE-1 cells de-creased as the cells were passaged. A similar loss of EBNA wasobserved in uncloned HONE-1 cells, but not in HONE-1 clone40 cells. In clone 40, which has been passaged 40 times thus far,85-90% of the cells are still EBNA-positive. The data suggestthat EBV genome-positive HNE-1 and HONE-1 cells were lostas the cells were cultivated in vitro and that cloning the cells atan early passage level may be critical in maintaining EBVgenome-positive epithelial NPC cells. These EBV genome-positive epithelial NPC cell lines will be useful for studying theassociation of EBV and NPC.

Epstein-Barr virus (EBV) is a human oncogenic herpesvirus.EBV has been associated with infectious mononucleosis (1),African Burkitt lymphoma (2), and nasopharyngeal carci-noma (NPC) (3, 4).

Initially, EBV was described as a B-lymphocyte-tropicvirus. The EBV DNA found in NPC tumors was presumed tobe in the infiltrating lymphocytes observed in such tumors. Ithas been shown, however, that EBV is actually associatedwith the epithelial cells of the tumor (5), particularly in theundifferentiated and partially differentiated, non-keratinizingforms of NPC (World Health Organization classes II and III)(6). Additionally, EBV DNA has been found in oropharyn-

geal epithelial cells obtained from normal individuals andfrom individuals with other, non-EBV-related oropharyngealtumors. These include normal and carcinomatous tonsillartissues (7, 8), tissues from the parotid gland (9), and biopsyspecimens from supraglottic laryngeal carcinomas (10); EBVDNA has also been found in nasopharyngeal biopsy samplesfrom normal individuals living in areas of China known to behigh-risk areas for NPC (11).

Epithelial cells of NPC tumors have receptors for EBV(12), as do normal nasopharyngeal cells from the nasophar-ynx of squirrel monkeys (13). In addition, it has been shownthat normal human epithelial cells can be infected with thevirus (14). Glaser and coworkers (15) demonstrated thattransformation of normal primary human epithelial cells,defined as the ability to grow in soft agar, resulted fromexposure of the cells to infectious EBV and that the trans-formation of these cells was dependent on the presence of aphorbol ester, phorbol 12-myristate 13-acetate. Establish-ment of long-term EBV genome-positive epithelial NPC celllines has not been reported. Often lymphoblastoid cell lines,but not epithelial cell lines, were established from biopsyspecimens of NPC. Only three permanent epithelial NPC celllines, CNE-1 and CNE-2 from NPC patients in China (16, 17)and NPC/HK1 from a patient in Hong Kong (18), have beensuccessfully established. Unfortunately, none of these celllines were confirmed to be EBV genome-positive after long-term culturing.

In this report we describe the HONE-1 and HNE-1 celllines, EBV genome-positive epithelial cell lines derived fromNPC tumors.

MATERIALS AND METHODSEstablishment of the HONE-1 and HNE-1 Cell Lines. A

portion of each NPC biopsy specimen was immediately putin RPMI 1640 medium (for HONE-1 cells) or a 1:1 mixture ofDulbecco's modified Eagle's medium and Ham's nutrientmixture F12 (for HNE-1 cells) (media were from Sigma).Approximately 1-mm3 tissue fragments were seeded into a25-ml glass tissue culture flask according to the methodroutinely used in our laboratory for the cultivation of humanembryonic nasopharyngeal epithelial cells (19). The cellswere incubated at 37°C and passaged twice per week.

Light and Electron Microscopic Examination. Cells forelectron microscopic examination were grown as a mono-layer and fixed in situ with phosphate-buffered 1.6% gluta-raldehyde containing 0.05M sucrose. Ultrathin sections werestained with uranyl acetate and lead citrate and then exam-

Abbreviations: EBV, Epstein-Barr virus; EBNA, EBV-encodednuclear antigen; EA, early antigen; VCA, virus capsid antigen; NPC,nasopharyngeal carcinoma.iTo whom reprint requests should be sent.

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ined using a JEOL or a Phillips 300 electron microscope.HONE-1 and HNE-1 cells were also grown as monolayers onglass coverslips, fixed in 70% ethanol, and stained withhematoxylin and eosin by routine procedures.

Southern Blot Analysis. Southern blot analysis was per-formed as described (20). In brief, DNA was extracted fromactively growing HONE-1 and HNE-1 cells. Restrictionenzyme digestion ofcellularDNA was carried out using 20 AgofDNA and 40 units ofBamHI restriction enzyme (IBI) in atotal volume of 30 Al. The EcoRI C fragment and the BamHIW fragment were labeled with [a-32P]dGTP and [a-32P]dCTP(ICN) by using a nick-translation kit (BRL) according to themanufacturer's recommendations.

Superinfection. Different subculture levels of HONE-1 andHNE-1 cells were superinfected with EBV. Three differentisolates of EBV were used, including the two prototypeisolates, B95-8 and HR-1, as well as an isolate derived froman NPC tumor, NPC-EBV (20, 21). Monolayer cultures wereprepared on glass coverslips. The cells were infected with

100x concentrates of filtered (0.45 ,um) virus as described(22). Cells were infected with each virus stock and allowed toincubate for 24, 48, or 96 hr. Cells were then washed inphosphate-buffered saline, fixed in acetone at room temper-ature, and examined for EBV-specific antigens by an indirectimmunofluorescence test (23) using early antigen (EA)/viruscapsid antigen (VCA)-positive human serum or monoclonalantibodies to EA-D (R3) and VCA (V3) obtained from GaryPearson (Georgetown University College of Medicine); atleast 1000 cells were scored.

Detection of the EBV-Encoded Nuclear Antigen (EBNA) inHONE-1 and HNE-1 Cells. HONE-1 and HNE-1 cells atdifferent passage levels were assayed for the presence ofEBNA. The anti-complement immunofluorescence test wasperformed on cell monolayers fixed in acetone/methanol(24). Previously characterized EBNA-positive human serumwas adsorbed on the cells, then human complement (derivedfrom an EBV negative donor) and goat anti-complementFITC (Meloy Laboratories) were adsorbed. All preparations

FIG. 1. (Upper) Hematoxylin/eosin-stained monolayer culture of HONE-1 cells. (x750.) (Lower) Electron photomicrograph of HONE-1cells fixed in situ. (x6015.)

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FIG. 2. Electron photomicrograph of HONE-1 cells showing two desmosomes between cells (arrows). (x21,000.)

were counterstained with 0.05% Evans blue and examinedwith a Zeiss microscope with an ultraviolet light source. Atleast 1000 cells were counted.

RESULTSClinicopathological History. The biopsy specimen from

which the HONE-1 cells were derived was obtained from a68-year-old Chinese man. The specimen from which theHNE-1 cell line was derived was obtained from a 27-year-oldChinese man. Both men were patients at the First AffiliatedHospital of Hunan Medical University. Both were diagnosedas having poorly differentiated NPC, and clinical examina-tion revealed tumor masses occupying the roof of the na-sopharynx, with left cervical lymph node involvement.

Establishment of the HONE-1 and HNE-1 Cell Lines. Epi-thelial cells with a few interposed fibroblasts grew as out-growths of small pieces of tissue after cultivation for 3 days.The cells could be passaged repeatedly. Generally, epithelial

cells grew steadily after cultivation in the flasks, when seededat 6.5 x 105 cells per flask; the cultured cells demonstratedloss of contact inhibition.

Histopathology of HONE-1 and HNE-1 Cells. A biopsyspecimen taken from each ofthe tumors for histopathologicalexamination showed poorly differentiated squamous cellcarcinomas. The HONE-1 cells in culture were found to bepoorly differentiated by light and electron microscopy (Fig.1). HNE-1 cells showed a similar morphology (data notshown). Desmosomes crossing cell membranes were ob-served in HONE-1 cells (Fig. 2) and HNE-1 cells (not shown)grown in culture, confirming the epithelial origin of the cells.

Detection ofEBNA in-HONE-1 and HNE-1 Cells. UnclonedHNE-1 cells were examined for the presence of EBNA atpassage level 12. Approximately 10% of HNE-1 cells werefound to be EBNA-positive (data not shown). By passage 35,it was no longer possible to detect EBNA-positive HNE-1cells.

FIG. 3. Immunofluorescence photomicrographs of HONE-1 clone 40 cells (passage 19), showing EBNA-positive cells. (x750.)

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FIG. 4. (A) Southern blot analysis of EBV DNA in HNE-1 cells.Cellular DNA was restricted with BamHI and hybridized with32P-labeled EBV BamHI W fragment. EBV genome-positive B95-8,HR-1, and NPC-KT cells and the EBV genome-negative HNE-3 cellswere used as controls. (B) Southern blot analysis of EBV DNA inHONE-1 and HNE-1 cells. CellularDNA was restricted with BamHIand hybridized with 32P-labeled EBV DNA EcoRI C fragment. TheB95-8, HR-1, and NPC-KT cells were used as controls.

Uncloned HONE-1 cells at passage 12 were found tocontain -18% EBNA-positive cells; at passage 23, -=3%; atpassage 35, -2%; and at passage 48, <1%; at passage 75 itwas not possible to detect any EBNA-positive cells. HONE-1clone 40 cells (which were cloned from HONE-1 parentalcells at passage 5) at passage 19, however, had 85-90%EBNA-positive cells, which has remained constant thus farup to passage 40 (Fig. 3).

Detection of EBV DNA in HONE-1 and HNE-1 Cells.HONE-1 and HNE-1 cells were assayed for EBV DNA atseveral different passage levels. Southern blotting detectedEBV DNA in uncloned HNE-1 cells at passages 12, 17, 21,27, and 35; in uncloned HONE-1 cells (passage 9), and so far

up to passage 40 in HONE-1 clone 40 cells. EBV genome-negative HNE-3 cells (another epithelial NPC cell line pre-pared in our laboratory) and EBV genome-positive B95-8,HR-1, and NPC-KT cells (21) were used as controls.

Initially, the HNE-1 cells were probed for the large internalrepeat present in theBamHI W fragment ofthe EBV genome.With this probe, prepared from B95-8 DNA, EBV DNA-positive cells were detected at passage 9 (Fig. 4A). In asecond set of experiments, the EcoRI C fragment was usedas a probe. The presence of EBV DNA was confirmed inHNE-1 with this probe (Fig. 4B). Preliminary data suggestthat the EBV genome in HNE-1 cells had the BamHI B'fragment but without the BamHI WI' fragment, unlike boththe HR-1 and the B95-8 isolates (Fig. 4B).When uncloned HONE-1 cells were assayed at passage 9,

using the same EcoRI C probe, EBV DNA was detected aswell (Fig. 4B). However, the EBV genome in HONE-1 cellsis similar to the B95-8 isolate on the basis ofBamHI mappingdata (Fig. 4B). More detailed mapping of the EBV genome inHONE-1 and HNE-1 cells is described elsewhere (25).

Ability to Superinfect HONE-1 and HNE-1 Cells with Infec-tious EBV. When uncloned HONE-1 cells at passages 17 and19 were exposed to different isolates of infectious EBV, wefound that the B95-8, NPC-EBV, and HR-1 isolates were ableto superinfect HONE-1 cells at a low level, as determined bythe detection of '=0.1% immunofluorescence-positive cells 48hr postinfection (Fig. 5). The percentage ofpositive cells thendecreased so that by 96 hr postinfection, none could bedetected. Uncloned HONE-1 cells at passages 47 and 68(which were EBV DNA- and EBNA-negative) could not besuperinfected with any EBV isolate.HNE-1 cells at passages 21 and 23 could be superinfected

with B95-8 and NPC-EBV, but not with the HR-1 isolate.When later passage uncloned EBV DNA- and EBNA-negative HNE-1 cells (over passage 100) were infected withvirus from the same virus stocks, no immunofluorescence-positive cells were observed for any of the virus isolates.

DISCUSSIONIt has been very difficult to grow NPC tumor cells intocontinuous cell lines. To our knowledge, only three epithelialNPC cell lines have been reported previously: CNE-1 (16),CNE-2 (17), and NPC/HKI (18). We have established three

FIG. 5. Immunofluorescence photomicrograph of HONE-1 cells (passage 17) superinfected with the HR-1 EBV isolate 48 hr postinfection.(x750.)

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epithelial NPC cell lines thus far: HONE-1 and HNE-1,which are EBV genome-positive, and HNE-3, which is EBVgenome-negative.

Cultivated uncloned HNE-1 and uncloned parentalHONE-1 cells have grown to passage levels >100 and >90,respectively. The morphology of HONE-1 and HNE-1 cells,as determined by light and electron microscopy, was that ofan epithelial tumor cell line, containing both undifferentiatedand differentiated epithelial cells. Desmosomes were com-monly observed in thin sections in HONE-1 and HNE-1cultures by electron microscopy.A small percentage ofHONE-1 and HNE-1 cells expressed

keratins (examined at passages 28 and 17, respectively),suggesting the presence of a small number of differentiatingcells. When HONE-1 and HNE-1 cells were inoculatedsubcutaneously in the subaxillary region of athymic nudeBALB/c (nu/nu) mice, tumors developed at the sites ofinoculation (25).The HNE-1 cells were examined for the presence of EBV

DNA by Southern blot analysis. The EBV genome wasdetected in cells at passages 12, 17, 21, 27, and 35 (data notshown). Preliminary Southern blot data suggest that the EBVgenome in HNE-1 cells had the BamHI B' fragment, butwithout the BamHI WI' fragment, in contrast to the B95-8and HR-1 isolates.By passage 35, it was very difficult to detect EBV DNA in

the uncloned HNE-1 cells with Southern blot analysis, and bypassage 80, no EBV DNA could be detected with thisprocedure. The loss of EBV DNA in HNE-1 cells occurredsimultaneously with the steady decrease in the percentage ofEBNA-positive cells.The BamHI restriction map of the EBV genome in the

HONE-1 cells suggests that the HONE-1 EBV genome issimilar to the B95-8 EBV genome. However, the EBVgenome in HONE-1 cells is different from both the B95-8 andthe HR-1 EBV genome in the HindIII map. A detailedrestriction map of the EBV genome from HNE-1 and HONE-1 cells is described elsewhere (25).We found that low-passage, EBV genome-positive un-

cloned HNE-1 and HONE-1 cells could be superinfected atlow levels with the B95-8 and NPC-EBV or the B95-8,NPC-EBV, and HR-1 isolates, respectively. Superinfectionof HNE-1 cells with HR-1 EBV did not result in the expres-sion of EA, suggesting either that this isolate was not able toinfect HNE-1 cells because the cells lack the appropriatereceptor or that the HR-1 EBV was not able to replicate inHNE-1 cells after penetration. Similar results were obtainedin an earlier study from our laboratory (26) in which it wassuggested that there may be differences in the receptors fordifferent EBV isolates.The EBV genome was lost (at least as determined by

Southern blotting) from the uncloned HNE-1 and HONE-1cells as the cells were passaged. This could be due to eitherthe loss ofEBV DNA from virus genome-positive cells or theloss of genome-positive cells as a result of our cultureconditions. The data suggest that EBV genome-positiveHNE-1 and HONE-1 cells were lost as the cells werecultivated in vitro and that cloning the cells at an earlypassage level may be critical in maintaining EBV genome-positive epithelial NPC cells. The data on the percentage ofEBNA-positive HONE-1 clone 40 cells at passages 19 and 40support this interpretation, since once we obtained HONE-1cells that were 85-90% EBNA-positive, the percentage ofEBNA-positive cells has remained stable through 40 pas-sages thus far. The data suggest that the in vitro growthconditions that we are employing may in some way select forEBV genome-negative cells.The ability to superinfect HONE-1 and HNE-1 cells was

lost by passage 68 and passage 100, respectively, suggestingthat the expression of the EBV receptor may have been

altered or masked as the cells were passaged in vitro or thatcells that were positive for the EBV receptor were selectedagainst as the cells were passaged. It is also possible that thepresence or expression of the EBV receptor on HONE-1 andHNE-1 cells may have been linked to the presence of thelatent EBV genome, since both markers were lost over time.The study of the relationship of EBV to NPC, and of the

process whereby epithelial cells are infected and trans-formed, has been hampered by the lack of an appropriate invitro model. The successful establishment of the HONE-1and HNE-1 cell lines will allow the interaction of the EBVgenome and epithelial (NPC tumor) cells to be studied underconditions that had not been possible before.Note Added in Proof. An experiment in which EBV genome-positiveHONE-1 clone 40 cells were mixed with EBV genome-negativeHONE-1 parental cells confirmed that the EBV genome-positivecells are selected against.

We thank Dr. Toru Takimoto for the NPC-KT cells from which weobtained NPC-EBV. We thank Ms. Kathleen Walker for the electronmicrographs. We thank the Department of Otorhinolaryngology,Hunan Medical University, for the NPC biopsy specimens.

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