FBJ Osteosarcoma Virus in Tissue Culture Isolation and Characterization of Non-Virus-Producing FBJ- Transformed Cells

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1 JOURNAL OF VIRoLOGY, Apr. 1978, p Vol. 26, No X/78/0026-O011$02.00/0 Copyright American Society for Microbiology Printed in U.S.A. III. FBJ Osteosarcoma Virus in Tissue Culture Isolation and Characterization of Non-Virus-Producing FBJ- Transformed Cells JAY A. LEVY,* PATRICIA L. KAZAN,' CHRISTOPHER A. REILLY, JR.,2 AND MIRIAM P. FINKEL2 Cancer Research Institute and Departnent ofmedicine, University of California, San Francisco, California 94143,1 and Division of Biological and Medical Research, Argonne National Laboratory, Argonne, IUinois Received for publication 28 October 1977 Hamster and rat cell lines have been established that have been transformed by FBJ murine sarcoma virus (FBJ-MuSV) but that do not produce virus. The hamster cell line originated from an osteosarcoma that appeared in a hamster inoculated at birth with an extract of a CF#1 mouse FBJ-osteosarcoma. The rat cell line was obtained by transferring the FBJ-MuSV genome to normal rat kidney cells in the absence of the FBJ type C virus (FBJ-MuLV), which, usually in high concentration, accompanies the FBJ-MuSV. Both transformed hamster and rat cell lines contain the FBJ-MuSV genome, which can be rescued by ecotropic and xenotropic murine type C viruses. This rescued genome produces characteristic FBJ-MuSV foci in tissue culture and, in appropriate animal hosts, induces osteosarcomas typical of those induced by FBJ-MuSV. FBJ-MuSV was isolated orginally from a parosteal osteosarcoma that occurred naturally in a mouse. Since there was no previous history of passage of the agent through any other animal species, these non-virus-producing hamstr and rat cells transformed by FBJ-MuSV should be very helpful in molecular studies examining the origin of spontaneous sarcoma genomes in mice. A naturally occurring murine sarcoma virus other MuSV, can transform secondary mouse (MuSV) was isolated by Finkel et al. from an embryo cells into autonomously replicating osteosarcoma that occurred spontaneously in transformed cells. This fact was demonstrated the thoracic spine of a male CF#1 mouse (5). by the inability of anti-fbj-musv antiserum to This sarcoma virus, named FBJ-MuSV, induces suppress focus formation in tissue culture cells parosteal osteosarcomas in mice after as short a after virus inoculation (12). Second, the FBJlatent period as 3 weeks (4, 6). Accompanying MuLV is present in such great excess (13) that FBJ-MuSV is another murine type C virus mouse cells can be easily infected by both FBJ (MuLV), designated FBJ-MuLV. This virus re- viruses, so titration patterns of FBJ-MuSV cansembles other MuLV by tissue culture studies, not be readily evaluated. including the inability to produce foci of cell The large amount of FBJ-MuLV accompaalteration in culture (12, 13). FBJ-MuLV has no nying FBJ-MuSV also makes it difficult to deknown pathological role in mice (12, 13). Both tect and quantitate FBJ-MuSV. Autoinhibition FBJ viruses are neutralized by antiserum against occurs (13): the associated FBJ-MuLV blocks AKR-MuLV and are N-tropic, since they pref- infection of cells by FBJ-MuSV so that extracts erentially infect N-type and not B-type mouse and tissue culture fluids must be diluted to demcells (12). In tissue culture, FBJ-MuSV produces onstrate the presence of FBJ-MuSV. Moreover, foci of cell alteration that are distinctly different the high concentration of FBJ-MuLV has prein both morphology and time of appearance (9 vented the isolation of non-virus-producing to 16 days) from foci induced by other strains of FBJ-MuSV-transformed cell lines. MuSV. FBJ-MuSV can transform rat cells in culture Although we suspect that FBJ-MuSV, like but so far has not induced tumors in rats. In other MuSV, is defective for replication, this contrast, hamster embryo cells in tissue culture question has not yet been adequately answered. resist infection and replication of the virus (12), The one-hit kinetics of focus formation observed but some hamsters inoculated with preparations in tissue culture with the virus (12) can be ex- of FBJ-MuSV have developed osteosarcomas (4, plained by certain properties of the FBJ-MuSV 5). In this paper, we evaluate the role of FBJpreparations. First, FBJ-MuSV, in contrast to MuSV in the induction of these hamster osteo- 11

2 12 LEVY ET AL. J. VIROL. sarcomas by characterizing one of them estab- cells and had a titer of PFU/ml. Rauscher-MuLV lished in tissue culture. and AKR-L1-MuLV were originally obtained from J. Hartley, NIAID, NIH, and were passed in NIH-Swiss- MATERIALS AND METHODS ME cells. NIH-Swiss-ME cells chronically infected with either of these viruses produced approximately Cell cultures. NIH Swiss and BALB/c mouse em- 1iO PFU of Rauscher-MuLV per ml and 106*3 PFU of bryo (ME) cells were purchased from Microbiological AKR-MuLV per ml. Assays of mouse tumors were Associates, Bethesda, Md. Hamster embryo cells were performed with 10% extracts as described previously obtained from Syrian hamster/anl fetuses. Human (12). Osteosarcoma induction was assayed by the inforeskin (HuF) cells, provided by Miriam Debby, Umr- jection of virus-containing materials into newborn versity of California, San Francisco, were used up to mice (4-6). passage 10, when their sensitivity to xenotropic virus Antiserum and neutralization assays. The diminished (11). preparation of rabbit antiserum to the NZB strain of The clone 1 cell line (derived from the NZB-Q xenotropic virus has been reported (14). Antisera to mouse cell line provided by Marilyn Lander, National AKR and Rauscher strains of ecotropic MuLV were Institutes of Health [NIH], Bethesda, Md.) has been obtained from J. Hartley. Neutralization tests were described (14). It produces approximately 103 infec- conducted as described earlier (14). Briefly, approxitious xenotropic virus particles per ml. The C57L-D mately 500 FFU of the various MuSV were mixed with cell line (dog sarcoma cells chronically infected with 2 to 4 neutralizing units of antiserum for 30 to 45 min the C57L xenotropic virus) was provided by P Arn- at room temperature. The mixtures were then diluted stein and J. Riggs, California Department of Health, 10-fold and added to tissue culture cells. Neutralization Berkeley (1). It produces about 105 infectious xeno- was considered to have occurred when focus formation tropic virus particles per ml. The normal rat kidney in the experimental cultures was one-third or less than cell line, NRK (3), was originally obtained from R. that in the control cultures, which had received only Ting, Biotech Research Laboratories, Rockville, Md. viru A clone of these cells susceptible to infection by both ecotropic and xenotropic MuLV was used. A line of RESULTS these NRK cells, nonproductively transformed by the Induction and isolation oftumors in ham- Harvey strain of MuSV (the NRK-Harvey line), was sters. Nine 1-day-old Syrian hamsters received established in our laboratory in San Francisco (9). We 0 *2-mlintra-abdominal CF. injection of l/anl also established an NIH-Swiss-ME line infected with a osteosarcomaexrct. of prepan FBJ-MuLV by making seven successive clonings by FBJ osteosarcoma extract. is preparation end point dilutions in NIH-Swiss-ME cells. XC cells, produced multiple osteosarcomas in CF#l/Anl a rat cell line transformed by but not producing Rous mnce within 2 to 3 months (Table 1A). The sarcoma virus, were provided by Wallace P. Rowe, hamsters were examined for tumor development National Institute of Allergy and Infectious Diseases by frequent palpation and roentgenography. (NIAID), NIH. Five of the nine died from natural causes; the Media. All cell lines were grown in Eagle minimal last death occurred 1,002 days after injection. essential medium supplemented with 10% fetal calf The other four developed osteosarcomas by 127, serum, 1% glutamine, and antibiotics (250 IU of peni- 175, 815, and 854 days. Fragments of tumor cillin per ml; 250 ug of streptomycin per ml). For ocus tissue from the animal that was sacrificed at 127 formation assays, the fetal calf serum was replaced dasweetrom the dorsal 127 with 5% heated (56 C, 30 min) calf serum. days were transplanted under the dorsal skin of Viruses and virus assays. For virus assay, al nine 15-day-old hamsters. After 28 days the cell lines were pretreated with DEAE-dextran to in- transplant from one of these was used to estabcrease their sensitivity to infection (2). Ecotropic lish the line of hamster tumor cells described (mouse-tropic) MuLV was detected by the XC plaque below. assay (17); titers are expressed as PFU per milliliter. Cultivation of the hamster tumor cell line Xenotropic virus was measured by cocultivation as- HT-FBJ. The hamster osteosarcoma described says as described previously (14). Briefly, fluids were above was minced and plated in tissue culture titrated on HuF cells. Inoculated monolayers were dishes with Eagle minimal essential medium as passed weekly for 3 weeks, and the cells were then desrbed Eag m of celld edin 5 cocultivated with NRK-Harvey cells. The titer of xen- described Small islands of cells developed in 5 otropic virus was estimated on the basis of pseudotype days, and within 2 weeks confluent monolayers virus formation and represented the end dilution of of tumor cells could be identified. This cell line the virus preparation that gave HuF the ability to (HT-FBJ) was passed weekly. Three clones of produce MuSV pseudotypes. Focus formation assays tumor cells, A-6, D-9, and F-6, were derived from for MuSV were conducted with mouse, human, and HT-FBJ by plating individual cells in microtest rat cells as described previously (14). Titers are ex- (Terasaki) dishes by techniques already depressed as focus-forming units (FFU) per milliliter. scribed (14) in spite of the difficulties imposed Stock FBJ virus preparations containing FBJ-MuSV and FBJ-MuLV were obtained from chronically infected NIH-Swiss-ME cell lines (12). The FBJ-MuSV Assays of hamster tumor cells for vipreparation had a titer of approximately 10o 5 FFU/ml ruses. Supernatant fluids from the parental by a plating efficency of less than5%c. and 107 PFU/ml. FBJ-MuLV was separated from hamster cell line, HT-FBJ, and the three cell FBJ-MuSV by end point dilution in NIH-Swiss-ME clones were assayed for focus-forming activity

3 VOL. 26, 1978 NON-VIRUS-PRODUCING FBJ-TRANSFORMED CELLS 13 TABLE 1. Induction of osteosarcomas in mice with FBJ-MuSVpreparations Mean no. of Virus No. of recip- Strain (range) A. FBJ-MuSV (FBJ-MuLV)a 12 CF#1/Anl 53.9 (41-91) 3.2 (2-5) 100 prepn no Virus Mean days to death osteosarcomas ~~~~ient mice (range) per mouse otsacma Miteosarcoma B. Pseudotype viruses FBJ-MuSV (Rauscher- 9 NIH-Swiss 31.1 (20-35) 2.7 (1-5) 100 MuLV)b 15 CF#1/Anl 37.1 (33-56) 2.6 (1-4) 100 FBJ-MuSV (Rauscher- 6 NIH-Swiss 29.5 (29-30) 2.2 (1-4) 100 MuLV) passage 2' 5 CF#1/Anl 29.4 (29-30) 3.6 (2-6) 100 FBJ-MuSV (AKR-MuLV)b 14 CF#1/Anl (37-238) 1.4 (1-3) 92 a Viruses were 0.45-,um-filtered, Hanks balanced salt solution extracts of tumor tissue. b Viruses grown in tissue culture. on mouse and rat cells. Monolayers of these cells TABLE 2. Rescue offbj-musvgenome from nonwere examined for ecotropic MuLV by the XC vru-producing transformed hamster cells plaque assay (17). No infectious virus was de- Foci/ml induced by tected. Culture fluids were tested for the pres- coculture supernaence of reverse transcriptase by techniques al- tant fluid in cells ready described (16), and enzyme activity was Cell line Cocultivated mono- fom: not observed. lrmice The parental cell line and the three clones (NIH- RRK were cocultivated with NIH-Swiss-ME and Swiss-ME) (K) NRK cells, and these mixed cultures were passed HT-FBJ NIH-Swiss-ME weekly for 3 weeks. No infectious virus was (Rauscher-MuLV) detected in the supernatant fluids from these C57l-Db 10 culture. NIH-Swiss-ME (FBJ MuLV) Rescue of the MuSV genome from ham- NIH-Swiss-ME - - ster tumor cells. The hamster tumor cell line HT-FBJ and the three clones derived from it Cell clones of were each cocultivated with NIH-Swiss-ME HT-FBJ NIH-SwiME 200 >500 cells chronically infected with either Rauscher- (Rauscher MuLV) MuLV, AKR-MuLV, or FBJ-MuLV. They were C57L-D - 10 also cocultivated with the NZB-Q clone 1 line D-9 NIHSsWssME 70 Nt and the C57L-D line. Control cultures included (Rau8cher-MuLV) the cell lines alone and cell lines cocultivated F-6 NIH-Swiss-ME (FBJ with non-virus-infected NIH-Swiss-ME cells. MuLV) Focus-forming virus was present in the super- (Rauscher-MuLV) 100 natant fluid of the cocultivation cultures when- C57LuD - 20 ever an MuLV was present in the added mono- NIH-Swiss-ME - layer cells (see Table 2 for representative exper- athe virus present in the cel lines used for cocultivation iments). Supernatant fluids from cultures con- is given in parentheses. tamning ecotropic virus induced foci in both ba dog cell line releasing the C57L zenotropic virus. mouse and rat cells, whereas fluids from cultures e-, No detectable virus. with murine xenotropic virus produced foci in dnt, Not tested. rat and HuF cells but not in mouse cells. The foci in NRK cells were typical of those previ- did not appear until 9 to 16 days after virus ously described for FBJ-MuSV and different inoculation. from those induced by standard MuSV (12). The The parental cell line (HT-FBJ) after coculfoci in HuF cultures could not be distinguished tivation yielded up to 3 logs of focus-forming from those induced in these human cells by virus per ml, as detected in NRK cells. The xenotropic pseudotypes of other MuSV (10). As clones, however, varied in their ability to express reported for conventional FBJ-MuSV (12), foci the viral genome. A-6 produced up to 3 logs of

4 14 LEVY ET AL. J. VIROL. focus-forming virus, D-9 yielded only about 1 TABLE 3. Neutralization ofmulvpseudotypes of log, and F-6 produced very few focus-forming FBJ-MuSV particles. The reason for this difference among FFU/ml in the clones is not known. The parental cell line, FBJ_ usvpseuotye_+_ntiera NRK cells when cocultivated with NIH-Swiss-ME cells FBJ (Rauscher-MuLV)a 42 chronically infected with the cloned FBJ-MuLV, + Anti-Rauscher-MuLV 0 yielded a virus preparation that titrated Anti-AKR-MuLV 40 FFU/ml in NRK cells and PFU/ml in NIH- Swiss-ME cultures. Cocultivation supernatant FBJ (NZB-MuLV)a 21 fluids containing focus-forming virus did not in- + Anti-Rauscher-MuLV 22 duce foci in hamster embryo cells. Anti-AKR-MuLV 20 Cultivation of rescued FBJ-MuSV vi- + Anti-NZB-MuLV 0 ruses. Some mouse and rat cell cultures that FBJ (Rauscher-MuLV)b 80 had been infected by an ecotropic MuLV pseu- + Anti-Rauscher-MuLV 3 dotype of FBJ-MuSV became completely trans- + Anti-AKR-MuLV 100 formed after three passages. Preparations of vi- a Virs was grown in tissue culture. ruses were obtained that titrated as high as 1036 b Virus was obtained from osteosarcoma extracts. FFU/ml and 1060 PFU/ml, as detected in mouse and rat cells. The various helper viruses were present at levels somewhat lower than the vast type of FBJ-MuSV was titrated on NRK cell excess found in standard FBJ preparations. monolayers. Those cultures with only one or two HuF cells infected with the NZB and C57L foci were maintained, and the foci were isolated xenotropic virus pseudotypes of FBJ-MuSV with a Pasteur pipette. This technique yielded were similarly passed until they appeared to be two NRK-FBJ cell lines of transfonned cells completely transformed. Preparations of the that did not release any focus-forming virus into xenotropic FBJ-MuSV titrated around 1020 the supernatant fluid. Upon superinfection by FFU/ml in both HuF and NRK cells. ecotropic MuLV or xenotropic MuLV, focus- Neutralization ofvirus isolates. The focus- forming virus with the envelope coat property of forming viruses rescued by Rauscher-MuLV, the helper MuLV was produced. These NRK AKR-MuLV, FBJ-MuLV, and xenotropic virus cell lines were subsequently cloned in Terasaki were examined by neutralization tests in tissue dishes, and cell lines from 10 individual cells culture to determine their envelope coat prop- were established. These 10 lines had properties erties. As shown in Table 3, each of the viruses similar to the parental non-virus-producing was appropriately neutralized by antiserum NRK cell lines. against the respective helper MuLV. Virus induction by IUdR. The parental Induction of osteosarcomas in mice. Dif- hamster cell line, HT-FBJ, was cultivated in the ferent preparations of the ecotropic virus pseu- presence of 30 u.lg of iododeoxyuridine (IUdR) dotypes of FBJ-MuSV were injected into new- per ml by standard techniques (15) in attempts born NIH Swiss and CFl#l/Anl mice. The re- to induce an FBJ-MuSV in a hamster type C sults of these studies are shown in Table 1B. A virus coat. Supernatant fluids from the IUdRtotal of 48 of the 49 mice inoculated with treated cultures were added to NIH-Swiss-ME, Rauscher or the AKR MuLV pseudotype of NRK, and hamster embryo cell cultures. No foci FBJ-MuSV developed at least one osteosar- of cell alteration were noted. However, similar coma. All tumors were typical of FBJ osteosar- experiments with the transformed NRK nonprocomas, regardless of the pseudotype virus used. ducer cell lines yielded low titers of a virus that Assay of virus from osteosarcomas in- induced foci only in NRK cells. Presumably, this duced in mice by the pseudotype FBJ vi- virus is a rat type C virus pseudotype of FBJruses. Extracts of the tumors induced in the MuSV. mice by the tissue culture-grown ecotropic virus DISCUSSION pseudotypes of FBJ-MuSV were assayed for focus-forming activity in tissue culture. In all This paper describes the viral characteristics cases, foci typical of FBJ-MuSV were induced. of an osteosarcoma that developed in a hamster These foci could be neutralized by appropriate inoculated with FBJ-MuSV. The data indicate antiserum against the original helper virus that that the cell line, HT-FBJ, cultivated from this formed the pseudotype MuSV (Table 3). tumor contains the FBJ-MuSV genome and Isolation of non-virus-producing rat strongly implicate this virus as the etiological celis. In attempts to transfer the FBJ-MuSV agent in these neoplasms. Tumor induction in genome to rat cells, a xenotropic virus pseudo- hamsters by sarcoma viruses has been a very

5 VOL. 26, 1978 NON-VIRUS-PRODUCING FBJ-TRANSFORMED CELLS 15 successful technique for generating non-virus- LrTERATURE Ci producing cells (7, 8). The HT-FBJ cell line 1. Arnstein, P., J. A. Levy, L S. Oshiro, P. J. Price, W. established in culture and the clones derived Suk, and E. IL Lennette Recovery of murine from it have all of the characteristics of other xenotropic type-c virus from C57L mice. J. Natl. Cancer non-virus-producing tumor cell lines, such as the 2.Due-Nguyen, Inst. 53: EL Ehnigeffect of diethylamino- HT-1 (8) and NRK-Harvey (9) lines. They do ethyl-dextran on the focus-forming titer of a murine not release any infectious virus as detected by sarcoma virus (Harvey strain). J. Virol. 2: biological and biochemical techniques. The sar- 3. Duc-Nguyen, EL, E. N. Rosenblum, and R. F. Zeigel. coma genome, is rescuable when the Persistent infection of a rat kidney cell line with coma genome, however, Rauscher murine leukemia virus. J. Bacteriol. cells are cocultivated with cells producing either 92: an ecotropic or xenotropic MuLV. The demon- 4. Finkel, M. P., and B. 0. Biskis Experimental stration that the FBJ-MuSV genome had been induction of osteosarcomas. Prog. Exp. Tumor Res. rescued was supported by the consistent produc- 10: tionypsedotye FBJMuSVof Vpicl 5.Finkel, KL P., B. 0. Biskis, and P. B. Jlnkinsi tion by pseudotype FBJ-MuSV (i) foci typical Virus induction of osteosarcoma in mice. Science of standard FBJ-MuSV in tissue culture and (i) 151: osteosarcomas in mice. These data indicate the 6. Finkel, M. P., P. B. Jinkins, J. Tolle, and B. 0. Biskis. stability of the FBJ-MuSV genome even after 16 Ser radiography of viu-induced osteosarco mas i integraion andsubseqent resue fromheter- numce. Radiology 87: integration and subsequent rescue 7. Huebner, R. J., D. Armstrong, KL Okuyan, P. S. ologous host cells. Sarma, and IL C. Turer Specific complement- By cocultivating the HT-FBJ line with cells fing vira antigens in hamster and guinea pig tumors producing xenotropic MuLV, a xenotropic virus induced by the Schmidt-Ruppin strain of avian sarpaeudotype of FBJ-MuSV coma. Proc. Natl. Acad. Sci. U.S.A. 51: pseudotype of FBJ-MuS5V was formed that read- 8. Huebner, R. J., J. W. Hartley, W. P. Rowe, W. T. ily permitted the transfer ofthe acoma genome Lane, and W. L Capps Rescue of the defective to NRK cells. From these cells, two non-virus- genome of Moloney sarcoma virus from a non-infectious producing lbj-musv-transformed NRK cell hamster tumor and the production of pseudotype sarlines were derived. These cell lines, NRK-FBJ, Poc. Nat. Aced. Sci. USA mu5n64 1uk69mia viru. and their clones have all of the biological char- 9. Levy, J. A Demonstation of differences in murine acteristics of HT-FBJ. Moreover, NRK-FBJ sarcoma virus foci formed in mouse and rat celis under cells can be induced with IlUdR to produce an a soft agar overlay. J. Natl. Cancer Inst. 46: infetiou.troi,..fbj-musv. that p. sm 10. I J. A Autoimmunity and neoplasia. The posinfectious rat-tropic trv-ntuo tnat presum- sible role of C-type viruses. Am. J. Clin. Pathol. ably carries the rat type C virus envelope. 62: Although the fact that hamster and rat cells 11. Levy, J. A Host range of murine xenotropic virus: fail to produce FBJ-MuSV suggests that the replication inaviancells. Nature (London) 263: virus is defective for replication, as are other Levy, J. A., J. W. Hartley, W. P. Rowe, and R. J. Huebner Studies of FBJ osteosamoma virus in MuSV, this question will not be completely re- tissue culture. I. Biologic characteristcs ofthe "C"-type solved until non-virus-producing FBJ-MuLV- virs. J. Natl. Cancer Inst. 51: transformed mouse cell lines are obtained. Com- 13. Levy, J. A., J. W. Hartley, W. P. Rowe, and IL J. Huebner Studies of FBJ osteosarcoma virus in petent Rous sarcoma viruses, for instance, do tissue culture. II. Autoinhibition of focus formation. J. not replicate in mammalian cells (8). These non- Natl. Cancer Inst. 54: virus-producing FBJ cell lines will permit an 14. Levy, J. A., P. L Kazan, 0. Varnier, and IL Klman. examination of the FBJ-MuSV genome free of Murine xenotropic type C virues.l. Distribution and the high concentration FBJ-MuLV, further characterization of the virus in NZB the high cncentratin of FBJ-MLV, which mice. J. Virol. 16: generally accompanies standard FBJ-MuSV 15. Lowy, D. R., W. P. Rowe, N. Teich, andj. W. Hartley. preparations Murine leukemia vius: high-frequency activation in vitro by 5-iododeoxyundine and 5-bromodeoxyuri- ACKNOWLEDGMENT:S dine. Science 174: WeWegratefiflyacassistancehe gratefully acknowledge the of Diane L Gutz- 16. Ross, J., E. K clnik G. J. Todaro, and S. A. Aaronson Separation of murine cellular and eit and Phylis J. Dale. murine leukaemia virus DNA polymerases. Nature This work was supported by Public Health Service grant (London) New Biol. 231: CA13086 from the National Cancer Institute and by the U.S 17. Rowe, W. P., W.. Pug1h, and J. W. ley Department of Energy. J.A.L. is the recipient of Public Health Plaque assay techniques for murnne leukemia viruses. Service Research Career Development Award 5KO4CA70990 Virology 42: from the National Cancer Institute.