Examination of Epstein-Barr Virus and C-Type Proviral Sequences in American and African Lymphomas and Derivative Cell Lines

[CANCER RESEARCH 41. 3165-3171, August 1981]

P. A. Pizzo,1 S. K. Chattopadhyay, I. T. Magrath, E. Del Ciacco, D. Sherrick, and T. Gray

Infectious Disease Section, Pediatrie Oncology Branch, National Cancer Institute, NIH, Bethesda, Maryland 20205

ABSTRACT these tumors are histologically indistinguishable (3, 57, 69). Even in Africa, less than one child in 1000 who is exposed to While Epstein-Barr viral (EBV) DMA is nearly always detect EBV at an early age eventually develops Burkitt's lymphoma able in African Burkitt's lymphoma (BL), the relatively low (15). Hence, the possibility that other factors operate in addi frequency of BL in EBV-positive African children and the infre tion to EBV to produce malignant transformation has been quent finding of EBV in American BL suggest that other cofac- raised. Among the possible cofactors which have been pro tors may contribute to the malignant transformation. posed are genetic defects [e.g., the 8; 14 translocation (32, 51 ) Among these possible cofactors are type C oncornaviruses. To and the X-linked lymphoproliferative syndrome (61)] or the evaluate this possibility, we screened the cellular DMA from 16 immunosuppression from holoendemic malaria which may per lymphomas (2 African, 14 American) and the DMA from 20 mit uncontrolled B-cell proliferation (16, 56). The possibility lymphoma-derived cell lines (4 African, 16 American) with a that specific strains of EBV may be oncogenic or that other radiolabeled viral DMA probe from EBV and two oncornaviral tumor viruses may serve as cofactors with EBV has also been probes (murine amphotropic 1504A virus and simian sarcoma suggested (25-34, 36). virus). The radiolabeled EBV DNA probe hybridized with 18 of 36 tumor or cell line DNA's. Only 2 of 11 American BL tumors Type C oncornaviruses have been described in a variety of subhuman species and tumors (20, 22). Moreover, some of contained detectable EBV sequences. However, the cell lines these oncornaviruses (MuLV, SiSV, gibbon ape leukemia virus, derived from three EBV-negative tumors converted in vitro to and baboon endogenous virus) have been associated with EBV positivity, suggesting that some of the tumor cells could human lymphoid cancers. This includes the finding of immature be infected with EBV. In contrast, none of the tumors or the intracytoplasmic type C particles with a sedimentation coeffi cell lines derived therefrom hybridized with either the 1504A cient of 550S, containing 70S RNA with homology to SiSV, or the simian sarcoma virus probes, decreasing the likelihood MuLV, and baboon endogenous virus by cDNA hybridization; that type C viruses are cofactors with EBV. the presence of SiSV and MuLV antibody-inhibitable reverse transcriptase; the electron microscopic demonstration of virus- INTRODUCTION like particles, and, less frequently, the presence of proviral DNA sequences (21, 22). In Burkitt's tumor tissue, Kufe ef al. Since its discovery in the cultured tumor cells from an African (41-43) described 70S particles containing RNA-dependent patient with BL,2 the EBV has become a leading candidate for DNA polymerase, and Kotler ef al. (40) observed the release of a human tumor virus (17, 18). This is based on several obser type C RNA virus particles from arginine-deprived human lym- vations. EBV DNA can be found in 97% of African Burkitt's phoblastoid cell lines which also contain EBV. tumors (55, 70), and 100% of these patients have significantly To further examine the possible association of oncornavi elevated serum antibodies to the viral capsid antigen and early ruses with human lymphomas, their derivative cell lines, and antigen of EBV (28, 30). Furthermore, in vitro infection with EBV, we have studied a variety of African and American lym EBV results in the transformation of human umbilical cord phomas and their cell lines for the presence of EBV and/or lymphocytes into continuous cell lines (24), and the inoculation oncornavirus proviral sequences. Two oncornavirus probes of EBV into owl monkeys and cotton-topped marmosets can were used. The amphotropic 1504A virus was selected as a result in a fatal lymphoproliferative disorder (13, 19, 66). While representative MuLV since at least 60% of its sequences are these properties of EBV are provocative, several unresolved homologous with the ecotropic and xenotropic viruses of lab dilemmas are also apparent with regard to the potential onco- oratory mice and 10% of its specific sequences (which are not genicity of this human virus. First, not only is EBV associated contained in the 60% of ecotropic and xenotropic sequences) with BL but it is also the causative agent of infectious mono- hybridize with the Friend, Rauscher, and Moloney leukemia nucleosis (29, 53). EBV has also been found in epithelial cells viruses (7, 8, 10, 26, 44, 47, 63). We also used a simian from patients with nasopharyngeal carcinoma. In contrast, EBV is found in only a minority of the cases of Burkitt's lymphoma sarcoma viral probe, since this virus has been associated previously with human leukemias and lymphomas (20-22). which occur sporadically outside equatorial Africa, although Together, these probes provide a broad screen for detecting oncornavirus sequences. ' To whom requests for reprints should be addressed, at National Cancer Institute, Building 10, Room 2B50, Bethesda, Md. 20205. 2 The abbreviations used are: BL, Burkitt's lymphoma; EBV, Epstein-Barr MATERIALS AND METHODS virus; MuLV, murine leukemia virus; SiSV, simian sarcoma virus; cDNA, comple mentary DNA; RPMI 1640, Roswell Park Memorial Institute Medium 1640; PCS, Tumors and Cell Lines. Fresh tumor material was divided fetal calf serum; TNE buffer, 0.05 M Tris (pH 8.0):0.1 M NaCI:0.001 M EDTA; into portions which were stored at â€”70Â°orwhich were finely SDS, sodium dodecyl sulfate; TE buffer, 0.01 M Tris (pH 7.6):0.001 M EDTA; PB, Phosphate buffer. minced, filtered through sterile gauze, and cultured in RPMI Received June 12, 1980; accepted March 5, 1981. 1640 plus 20% FCS (Grand Island Biological Co., Grand Island,

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N. Y.) at 5% CO2. The cell lines which have been established RD cells which were chronically infected with 1504A virus were maintained and propagated in McCoy's Medium 5A (Grand from many of these tumors were maintained as suspension cultures at 37Â° in 75-sq cm plastic flasks and subcultured Island Biological Co., Grand Island, N. Y.) with 10% heat- every 3 to 4 days. The growth characteristics and surface inactivated FCS. Cells were seeded in 150-sq cm plastic flasks properties of many of these derived cell lines have been ana (Corning Products, Corning, N. Y.); when confluent, the super lyzed recently (45, 46). For the preparation of DMA from these natant (20 ml/flask) was collected at 12-hr intervals, centri cell lines, approximately 5 liters of each cell line were cultured fuged at 9150 x g for 10 min at 4Â°,decanted into a sterile 20- in RPMI 1640 plus 10% PCS for 4 days, at which time the cell liter carboy, and stored at 4Â°.The virus was concentrated by viability exceeded 90%. Cells were pelleted by low-speed in a 20 to 50% sucrose gradient in a Model K ultracentrifuge centrifugation and washed 3 times with 0.85% phosphate- (Electronucleonics Corp.). The virus-containing fractions (mea buffered saline (0.0067 M Na2PO4, 0.0067 M KH2PO4, and sured by absorbance at A28o)were banded a second time in a 0.85% NaCI solution, pH 7.2). Cell pellets and tumors were 20 to 50% sucrose density gradient in a CF-32 rotor (Beckman stored at -70Â° until used. Instrument Co., Palo Alto, Calif.). The virus-containing fractions Preparation of Cellular DMA. Unlabeled DNA was extracted were then pooled, pelleted at 99,000 x g, resuspended in 0.1 from both tumor tissue and cell pellets. Tumor tissue was M NaCI:0.01 M Tris (pH 7.4):0.001 M EDTA, and stored at minced, suspended in TNE buffer, and homogenized in a -70Â°. Waring Blendor and tissue grinder. DNA extraction was then SiSV-1 virus was prepared together with woolly monkey performed according to a previously described method (52) leukemia virus in a manner similar to that for the NC-37 cell which included detergent lysis with SDS; enzymatic digestion line. The 3H-SiSV-1-cDNA probe used in this study was pro of protein and RNA with nuclease-free Pronase (Calbiochem, vided from Drs. D. Lowy and E. M. Scolnick (National Cancer San Diego, Calif.; predigested for 1 hr at 37Â°), pancreatic Institute, Bethesda, Md.). The propagation of the virus, synthe RNase (XIIA; Sigma Chemical Co., St. Louis, Mo.), RNase Ti sis of [3H]cDNA, and its characterization have been described (Calbiochem, B grade), and subtilisin (Nagarse; Enzyme De elsewhere (64, 65). velopment Co., Osaka, Japan); extraction with TNE buffer- Synthesis of Single-stranded, Virus-specific [3H]DNA. Sin saturated freshly distilled phenol; and ethanol precipitation. gle-stranded virus-specific [3H]DNA was synthesized in an en The DNA extracted by this method had a A26o:A28oratio of 1.90 dogenous reverse transcriptase reaction using detergent-lysed to 1.95. purified virions in the presence of actinomycin D. The synthe Preparation of Radiolabeled Viral DNA Probes. EBV was sized probe was purified by deproteinization with chloroform: purified from the AG876 cell line according to a previously isoamyl alcohol (24:1, v/v) and phenol, and unincorporated described method (58). In brief, AG876 cells were cultured in radioactivity was removed by Sephadex G-50 (Pharmacia Fine RPMI 1640 plus 10% PCS at 32-33Â° and were labeled on the Chemicals, Inc., Piscataway, N. J.) chromatography. RNA was eighth day of culture with [3H]thymidine (Amersham/Searle, removed by alkali treatment followed by neutralization and Arlington Heights, III.; 45 Ci/mmol) at a concentration of 5 /Â¿Ci/ dialysis. The 3H-viral probe synthesized by this method had a ml. After 14 days of continuous culture, cellular debris was specific activity of 2 x 107 cpm//Â¿g and was 98% single- pelleted by low-speed centrifugation, and the supernatant was stranded and about 200 to 400 nucleotides long. More than filtered through a 1-/im Millipore filter (RATF 14200; Millipore 90% of the probe sequences are present in the viral RNA (11 ). Corp., Bedford, Mass.), concentrated by ultrafiltration through Hybridization Reactions. The unlabeled DNA extracted from a Millipore Pellican cassette system with a nominal molecular tumors and cell packs and the 3H-labeled EBV DNA probes weight cutoff of 1 x 106 to 3 x 106. The supernatant viral were each sheared twice by passage through a French pres concentrate was pelleted by ultracentrifugation (113,000 x g sure cell (American Instrument Co., Silver Spring, Md.) with a for 1 hr at 4Â°)and resuspended in 3 ml of TNE buffer. Virus pressure drop of 40,000 psi. Sheared DNA's were each passed was purified by dextran density sedimentation (dextran, M.W. through a Metricel GA-6 filter (cellulose acetate; Gelman In 10,000; p 1.04 to 1.09 g/ml; 81,500 X g for 70 min at 4Â°). strument Co., Ann Arbor, Mich.), extracted twice with chloro- Radiolabeled virus-containing peaks were pooled, diluted with form:isoamyl alcohol, and precipitated with ethanol. TE buffer, and purified by isopyknic sucrose banding (30 to Hybridization reactions with the radiolabeled EBV DNA probe 60%, w/v); and the radioactive peak was pelleted and dialyzed consisted of sheared unlabeled tumor or cell line DNA (5.6 mg/ml) mixed with 7 x 10~2 jug of 3H-AG876 EBV DNA in a 1- against TE buffer. The purified virions were then lysed with SDS and sedimented through a 10 to 30% (w/v) sucrose ml Reactivial (Pierce Chemical Co., Rockford, III.). The mixture gradient (198,000 x g for 3 hr at 20Â°),and the 55S radioactive was denatured by heating to 108Â°for 5 min in 0.12 M PB which viral DNA peak was pooled, dialyzed against TE buffer, and was then adjusted to 0.48 M PB. The mixture was incubated at then extracted twice with TNE-saturated phenol and with chlo 65Â°, and aliquots were removed at different time points and roform :isoamyI alcohol (24:1, v/v). The aqueous phase was diluted to 2 ml with a final concentration of 0.14 M PB plus precipitated with cold ethanol (95%), and the precipitate was 0.4% SDS. The percentage of hybridization was determined by resuspended in TE buffer and then centrifuged to equilibrium hydroxyapatite chromatography (Bio-Gel HTP; Bio-Rad Labo in cesium chloride (129,000 x g for 60 hr at 20Â°).The single ratories, Richmond, Calif.), unhybridized molecules being radioactive peak at a density of 1.720 g/ml was pooled, eluted from the columns at 60Â°with 8 ml of 0.14 M PB plus dialyzed against TE buffer, and determined to have a spectro- 0.4% SDS and hybridized molecules being eluted with 8 ml of photometric A260:A280ratio of 1.90 and a specific activity of 0.32 M PB. Twelve ml of Instagel (Packard Instrument Co., 50,000 cpm/fig. Downers Grove, III.) were added to each for liquid scintillation The amphotropic 1504 virus (1504A) used in this study was counting. A minimum of 12 to 15 samples were assayed for grown in a human rhabdomyosarcoma (RD11 4) cell lines (47). each reaction mixture during a 72-hr incubation, and the per-

3166 CANCER RESEARCH VOL. 41

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1981 American Association for Cancer Research. EÃŸVand C-Type Proviral Sequences in Lymphomas centage of hybridization of viral DMA was analyzed as a func tion of C0f (the product of the viral DNA concentration in the reaction mixture and the duration of the hybridization reaction, calculated as nucleic acid absorbance at 260 nm times the duration of incubation in hr divided by 2). In hybridization reactions with the radiolabeled oncornaviral cDNA, sheared DNA (10 mg/ml) from tumor or tissue culture cells was mixed with 1 x 10~3 /ig of the amphotropic 1504A MuLV cDNA probe or with 1 x 10~3 fig of the SiSV-1 cDNA probe in 0.3-ml Reactivials. The mixtures were denatured in 0.14 M PB by heating to 100Â° for 4 min, adjusting to 0.48 M PB, and incubating at 65Â°. A minimum of 8 samples were removed from each reaction mixture during the subsequent 72 hr of incubation. Hybridization was again determined by hydroxyapatite chromatography, with unhybridized molecules being eluted from the column with 8 ml of 0.14 M PB plus 0.4% SDS at 60Â°.Hybridized molecules were eluted from the column at 99Â°with 8 ml of 0.14 M PB plus 0.4% SDS. The percentage of hybridization of the unlabeled cell DNA was measured in each sample by the spectrophotometric absorbance at 260 nm. Following this, 12 ml of Instagel were added to each sample, and the percentage of hybridization of the labeled viral DNA probe was measured by liquid scintillation counting.

RESULTS Thirty-six cellular DNA's from lymphoma patients were stud ied, 16 tumors (2 African, 14 American) and 20 cell lines (4 African, 16 American). In 11 cases, both the original lymphoma tissue and its derivative cell line were available for study. All 36 cellular DNA's were studied with the radiolabeled EBV probe. Thirty-three of the 36 cell DNA's were also studied with the amphotropic MuLV and SiSV-1 viral cDNA probes; insuffi cient quantities of 3 tumor DNA's were available for testing with the amphotropic MuLV and SiSV-1 probes. In addition, cellular DNA's from human placental tissue, from a human rhabdomyosarcoma tumor, and from RD114 cells infected with ampho tropic MuLV served as controls. Hybridization with 'H-EBV DNA. Eighteen of the 36 unla beled DNA's from tumor tissue culture cell lines contained detectable EBV DNA sequences. Three patterns of hybridiza tion with 3H-AG876 EBV DNA were observed. The first was complete hybridization (i.e., >90%) and was demonstrated in 6 of the 18 DNA's (Chart 1/\). Four of these DNA's were from EBV producer cell lines (P3HRI, B95-8, JLP, and AG876). A second pattern of hybridization, in which the radiolabeled EBV probe hybridized from 81 to 89%, was observed with 5 of 18 cell DNA's (Chart 1B). Hybridization with EBV was incomplete (62 to 80%) with 7 cellular DNA's (Chart 1C), one of these cell lines being a virus producer. The remaining 18 DNA's showed 10' 10J 101 10' no discernible hybridization with EBV [3H]DNA (Chart 1-4). CELLULAR DNA Cot (moles x seconds/liter) Each of the 6 DNA's of African origin (2 tumors, 4 cell lines) Chart 1. Hybridization kinetics of radiolabeled viral probes with unlabeled cellular DNA. A 3H-EBV DNA probe with human placental DNA (Â») and 3 hybridized to between 89 and 94% with the 3H-AG876 EBV representative EBV-negative cell DNA's. D, Conception cell line; *. Wilson tumor; probe. Of the 14 lymphoma tumor samples of American origin < . Thomas cell line; , JL pleural cell line; â€¢RAJI cell line; A. B95-B cell line; (9 Burkitt's, 3 undifferentiated, and 2 lymphoblastic lympho- â€¢,AG876 cell line; O, MS115 cell line; A, P3HRI cell line. B, â€¢ â€¢,3H-EBV mas), 2 (both Burkitt's) hybridized with 3H-EBV (one to 62% DNA with human placental DNA; D. TE cell line; A, BHT bone marrow cell line; Â».KK124 cell line; â€¢.JL ascites; â€¢ASB cell line; A. BHT ascites cell line; O, and the second to 86%). Of the 16 cell lines derived from DW36 cell line. C, Â».3H-EBV DNA with human placental DNA; A, AMP ascites; â€¢ABANA ascites; O. AG876 tumor; A. MOY tumor; â€¢BR tumor. D, A, 1504A American lymphoma tissue, 9 were EBV genome positive (62 3H-DNA with RD cell DNA; â€¢1504A [3H]DNA with human placental DNA; Â». to 93% hybridization) while 7 contained no detectable EBV 1504 3H-DNA with 1504A-infected RD cell DNA; O O, RD cell DNA self- sequences (Table 1). association kinetics; D, 1504-A [3H]DNA with AG876 cell DNA; O O, 1504- A [3H]DNA with PD-2 cell DNA; A, 1504-A [3H]DNA with KK tumor DNA; dotted The original tumor and its derivative cell line were available open triangles. SiSV [3H]DNA with P3HRI cell DNA; dotted open diamonds. SiSV for hybridization with EBV in 11 cases. One of these tumors 3H-DNA with RAJI DNA; grouped closed triangles, SiSV DNA with KK tumor DNA.

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Table 1 Percentage of hybridization of the radiolabeled E8V. MuLV, and SiSV- i viral DNA probes with tumor and derived cell line cellular DNA

ofhybridizationEBV8489949395949286899218181614252222252522222222222222811675629222228062806422Ampho some 8; 14 ab Cellular normali DNAAbanaAG876AG876MS115B95-8P3HRIRAJIMOYAMPBRCA-46CA-46EW-36EW-36ST486ST486VVMC116MC116PD2PD2TRGMCSTRDWDW6KKKK124JLJLJLJAJATEBHTBHTRD114D188OriginAfricanAfricanAfricanAmericanAmericanAfricanAfricanAmericanAfricanAmericanAmericanAmericanAmericanAmericanAmericanAmericanAmericanAmericanAmericanAmericanAmericanAmericanAmericanAmericanAmericanAmericanAmericanAmericanAmericanAmericanAmericanAmericanAmericanAmericanAmericanAmericanAmericanAmericanAmericanMistologicaldiagnosisBLBLBLBLInfectious ties8;14NA8;14OtherNA14qtropic8.4NA8.710.09.57.43.07.96.04.411.69.0NA9.05.46.0NA3.75.06.09.57.46.39.55.012.14.65.07.07.16.07.06.05.08.95.08.071SiSV-16.0NA5.07.06.06.07.06.05.06.06.06.0NA5.04.05.0NA5.06.05.05.05.06.09.05.06.05.05.06.06.05.07.05.05.06.07.07.0 (CL)aSplenic (T)Splenictumor (CL)Ascitestumor (CL)Peripheral mononucleosisBLBLBLBLRhabdomyosarcomaBLBLUndifferentiatedUndifferentiatedBLBLBLBLUndifferentiatedUndifferentiatedLymphoblasticLymphoblasticBLLymphoblasticBLBLBLBLBLBLBLBLBLBLBLUndifferentiatedUndifferentiatedSarcomaHuman(CL)Tumor blood (CL)Tumor +14q (CL)Tumor +NANANA8:148;1414q14q14q14qNANANA8;148;148;14NANANA8;148;14OtherOther8;148;14OtherNANANA8;148;14NA% (T)Ascites (T)Tumor (T)Ascites (T)Ascites (CL)Pleural (T)Pleuraleffusion (CL)Asciteseffusion (T)Ascites (CL)Tumor (T)Tumor (CL)Pleural (T)Pleuraleffusion (CL)Pleuraleffusion (T)Pleuraleffusion (CL)Tumoreffusion (T)Pleural (T)Pleuraleffusion (T)Pleuraleffusion (T)Pleuraleffusion (CL)Asciteseffusion (T)Ascites (CL)Ascites (T)Pleural (T)Bone fluid (CL)Tumormarrow (T)Tumor (CL)Bone (CL)Ascitesmarrow (CL)Bone (CL)Amphotropicmarrow (CL)Normal infected placentaSourceAscites tissueChromo CL, cell line; T. tumor, NA, cytogenetics not available; other, chromosome markers other than No. 8 or No. 14 present. was of African origin (AG876), and the 3H-EBV DNA probe EBV genome positivity. hybridized to >90% with the DNA from both the tumor tissue Hybridization with C-Type Probes. Reaction of the 3H-am- and its derived cell line (Table 1). The other 10 tumor cell line photropic 1504A-MuLV cDNA probe with the unlabeled DNA pairs were of American origin. With one pair (JL), the 3H-EBV extracted from the amphotropic virus-infected RD114 cells DNA probe hybridized with both the tumor and its derived cell hybridized to 90% (Chart 1D). However, both the amphotropic lines. The remaining tumors contained no detectable EBV and SiSV-1 cDNA probe failed to hybridize with any of the 33 tumor or derived cell line DNA's or with human placental DNA sequences, and in 6 cases the derived cell line was similarly negative for EBV. In 3 cases, however, the original tumor was or rhabdomyosarcoma DNA. The kinetic patterns of these EBV negative, but the derived cell line contained detectable reactions were virtually identical for all tumor and cell line DNA's. Representative patterns are shown in Chart 1D for 6 EBV sequences. In one of these cases (JA), the derived cell lines appeared to be diploid and probably represented the such hybridization reactions. In each of these reactions, spec- overgrowth of a normal EBV lymphoblastoid cell line. In the trophotometric analysis confirmed that the cellular DNA self- other 2 cases (DW and KK), however, the cell lines were hybridization had progressed to completion. aneuploid and had karyotypes consistent with that of the orig inal tumor (including an 8; 14 translocation for the DW and DISCUSSION DW6) and hence were considered to be tumor derived. One of these, cell line KK124, was established from tumor ascites in Serological and biochemical evidence for the association of a patient with American BL who had relapsed shortly after a EBV with African BL can be found in approximately 97% of the serologically documented episode of infectious mononucleo tumors and their derivative cell lines (17, 18, 28, 30, 55, 70). sis. It is notable that the tumor samples obtained from this In contrast, a similar association with EBV is available in only patient at the time of initial diagnosis and after infectious 9 to 17% of the histologically indistinguishable American BL mononucleosis were EBV genome negative. It is possible that (3, 57, 69). Whether there are 2 types of BL (one associated some of the tumor cells (too few for detection by hybridization) with EBV and the other not) remains unresolved. In addition, obtained at the time of relapse had been converted in vivo to whether additional factors contribute to or enhance the onco-

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1981 American Association for Cancer Research. ESV and C-Type Proviral Sequences in Lymphomas genie potential of EBV is not known. The known association of probe because of its previous association with human leuke EBV with a nonmalignant disease (infectious mononucleosis) mia, although proviral sequences have been generally less (29, 53) further confounds an understanding of the oncogen- detectable. The murine amphotropic viral probe was selected icity of EBV. since it shares 80% homology with the Rauscher and Moloney The evidence to date also suggests that there is no apparent leukemia viruses which have also been detected in human difference in the linear integrated or high-molecular-weight lymphoid tumors. Consequently, we hypothesized that the am circular episomal forms of EBV in African or American BL (1, photropic cDNA would permit an initial screening which could 2, 4, 6, 35, 36, 39). Furthermore, consistent differences have be more specifically explored with selected oncornaviruses. also not been demonstrated in the circular DNA's found in The lack of proviral sequences in this study does not exclude nontumorous EBV-infected lymphoblastoid cell lines in com the possibility that 70S RNA or RNA-dependent DNA polym- parison with African BL. Whether different strains of EBV or erase might be present, nor can we exclude the possibility that different sites of integration are related to the oncogenic a C-type virus had previously entered the "tumor" cell, con expression of this virus is also unknown but under study (23, tributed to its transformation, and was then excised and ren 25, 27, 37, 48, 50, 58, 60). dered undetectable (20-22). Our hybridization technique In the present series, we were able to demonstrate detectable would also fail to detect the presence of small fragments of EBV in only 2 of the 11(18%) American BL tumors examined. oncornavirus. Two additional non-Burkitt's American lymphoma tissues were It is of additional interest that the tumor-derived cell lines examined and were found to lack EBV sequences. Ten Amer also failed to reveal the presence of proviral sequences. It is ican lymphoma tumors gave rise to lymphoid cell lines. In 6 well established that prolonged tissue culture may permit the cases, both the original tumor and derived cell line were neg expression of virus activity not demonstrated in the original ative, while in one case the tumor and cell line were positive. tumor (33, 34, 68). On the other hand, while tissue culture may However, EBNA staining showed that only a small fraction of enhance type C virus replication, it may also permit the labo this cell line, when first derived, contained EBV. This rapidly ratory contamination of cell lines by oncornaviruses. Finally, it increased to 100%. The remaining 3 original tumors were EBV should be noted that, in addition to reports finding oncornaviral negative, while the derived cell line was positive. All of these material in human leukemias and lymphomas, negative studies cell lines have an aneuploid karyotype consistent with the have also been reported (9, 38, 52). original and 2 had 8; 14 translocation, which suggests that they Thus, the majority of the American lymphomas that we ex are tumor-derived cell lines. In one of these (DW), the conver amined lack both EBV and type C viral sequences. The lack of sion to EBV positivity occurred in vitro and is unexplained. In association of these tumors with EBV and the observation that the case of KK, the original tumor was EBV negative. However, some of the negative tumor cells can be infected with EBV several months following the initial diagnosis, the patient de suggest that it is unlikely that this virus is a causative factor in veloped serologically documented infectious mononucleosis American BL. Furthermore, it might be concluded either that and then relapsed. A tumor cell line established from the ascitic the histologically identical EBV-positive African BL is truly a fluid at the time of relapse was EBV positive. These findings separate disease in which EBV plays an important etiological suggest that, while EBV was not detected in the original tumor, role or that EBV is simply resident in a cell which undergoes at least some of the tumor cells could have been infected by transformation by some other process. The contribution of EBV. While these observations seem to further minimize the oncornaviruses in this transformation process is not substan role of EBV in American BL, our data do not permit us to tiated by this study. exclude the possibility that small portions or fragments of the EBV genome were present (but currently undetectable) in the apparently genome negative DNA's. In this regard, the partial REFERENCES

hybridization of the EBV probe with some of the cell DNA's is 1. Adams. A.. Bjursell, G.. Kaschka-Dierich, C., and Lindahl, T. Circular Ep- stein-Barr virus genomes of reduced size in a human lymphoid cell line of of interest and may reflect that only a portion of the EBV infectious mononucleosis origin. J. Virol., 22: 373-380, 1977. genome is present (59). 2. Adams, A., and Lindahl, T. Epstein-Barr virus genomes with properties of It is notable that the human rhabdomyosarcoma tumor se DNA molecules in carrier cells. Proc. Nati. Acad. Sei. U. S. A., 72: 1477- lected as a control contained detectable EBV sequences. While 1481, 1975. 3. Andersson, M., Klein, G., Ziegler, J. L., and Henle, W. Association of the most likely explanation is that this was due to infiltration of Epstein-Barr viral genomes with American Burkitt's lymphoma. Nature the sarcomatous tissue by EBV-positive lymphocytes (31), it (Lond.), 260: 357-359, 1976. 4. Andersson, M., and Lindahl, T. Epstein-Barr virus DNA in human cell lines: remains possible that the rhabdomyoblasts contained the viral in vitro conversion. Virology, 73. 96-105, 1976. sequences. Since it is known that the epithelial cells found in 5. Andersson-Anvret, M., Forsby. M., and Klein, G. Nasopharyngeal carcinoma. nasopharyngeal carcinoma can contain EBV, a precedent for Prog. Exp. Tumor Res., 21: 100-116, 1978. 6. Andersson-Anvret, M., and Lindahl, T. Integrated viral DNA sequences in nonlymphoid EBV infection is available. Epstein-Barr virus DNA in an American Burkitt's lymphoma line. J. Virol., We were surprised by the absence of any detectable homol- 25. 710-718, 1978. ogy with the 2 oncornavirus probes used in this study. Since 7. Aulakah, G. S., and Gallo, R. C. Rauscher leukemia virus related sequences in human DNA: presence in some tissues of some patients with hematopoietic previous workers have described oncornavirus-like particles in neoplasias and absence in DNA from other tissues. Proc. Nati. Acad. Sei. U. at least 2 of the cell lines that we examined (RAJI and P3HRI), S. A., 74:353-357, 1977. 8. Baxt, W. Sequences present in both human leukemia cell nuclear DNA and our negative findings are in direct conflict. Several possibilities Rauscher leukemia virus. Proc. Nati. Acad. Sei. U. S. A., 71: 2853-2857, might explain the lack of detectable proviral DNA sequences in 1974. this study. First, the probe(s) that we selected might have 9. Bernard, C., Chant, J. C., and Olivie, M. Search for endogenous C-type viruses in cultures of nonleukemic human cells. Biomedicine, 29: 143-146, simply missed or failed to detect resident oncornaviral se 1978. quences. While this cannot be excluded, we chose the SiSV-1 10. Chattopadhyay, S. D., Hartley, J. W.. Lander, M. R., Kramer, B. S., and

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