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[CANCER RESEARCH 42, 2523-2531, July 1982] 0008-5472/82/0042-OOOOS02.00 Growth of Murine Sarcoma and Murine Xenotropic Leukemia Viruses in Japanese Quail: Induction of Tumors and Development of Continuous Tumor Cell Lines W. G. Robey,1 W. J. Kuenzel, G. F. Vande Woude, and P. J. Fischinger Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick, Maryland 21701 [W. G. R., P. J. F.]; Department of Poultry Science, University of Maryland, College Park, Maryland 20724 [W. J. K.]; and Laboratory of Molecular Virology, National Cancer Institute, Bethesda, Maryland 20205 ¡G.F. V. W.] ABSTRACT and induces sarcomas in mice, rats, and hamsters (19, 32). Most MSV isolates do not have a functional reverse transcrip- The BALB/c murine endogenous xenotropic leukemia virus tase (31) or envelope glycoprotein (34, 36) or the genetic pseudotype of ml Moloney sarcoma virus [mlMSV(B-MuX)] information to specify those polypeptides (9, 39). In order to was used to productively transform diploid Japanese quail replicate, MSV isolates must obtain these functions from any embryo cells. The majority of newly hatched quail inoculated number of replicating helper viruses from various species (20, with ml MSV(B-MuX)-transformed quail embryo fibroblast cells 31 ). ml MSV (13) is a defective retrovirus containing nucleotide rapidly developed tumors which were predominantly locally sequences derived from M-MuLV (~3.8 kilobases) and normal invasive fibrosarcomas, but métastaseswere observed in two mouse cellular sequences, mos (~1.4 kilobases) (16, 39, 40). birds. In two tumor-bearing quail, lymphosarcomas were ob The mIMSV does express a portion of the leukemia virus served in conjunction with fibrosarcomas. Quail inoculated with group-specific antigen gene (gag) in the form of an aberrantly ml MSV(B-MuX) virus or quail embryo fibroblast cells infected cleaved polyprotein, pP60aas (29, 30, 34). It has been shown with helper leukemia virus did not develop tumors. A cell culture with cloned mIMSV proviral DNA that the essential nucleotide derived from one quail tumor was shown to be oncogenic in sequences for transformation by mIMSV reside in the long newly hatched quail. terminal repeat and a region of the genome containing mos (4, Viruses produced by m1MSV(B-MuX)-infected quail cells or 27). Unlike the ASVs, a translation product of mos sequences quail tumors had envelope properties of BALB-derived murine required for the maintenance of transformation has not been xenotropic leukemia virus as measured by host range, interfer identified for mIMSV. In the avian system, a nonstructural ence, and neutralization. Virus structural antigens, proteins phosphoprotein with phosphokinase activity (pp60src) has been with molecular weights of 30,000 and 70,000, were detected determined to be the translational product of the ASV src which in tumors and tumor-derived cell lines by immunofluorescence is associated with maintenance of transformation (3, 5, 7). The and gel diffusion. Sera from tumored quail had high titers of avian pp60src was identified by immunoprecipitation with sera type-specific BALB-derived murine xenotropic leukemia virus from rabbits which rejected ASV tumors. Because mouse antibodies as determined by neutralization and ¡mmunoprecip- helper viruses from the MuX group could replicate in avian itation. Antibodies to the putative ml Moloney sarcoma virus cells (26) and because extensive evolutionary divergence may mos gene product were not detected in sera from tumored or be required to produce antibody to the murine mos gene régresserquail. product, we induced regressor tumors in birds with ml MSV(B- MuX). Quail proved to be an amenable animal in which tumors INTRODUCTION could be induced at high frequency with diploid quail embryo cells infected by ml MSV(B-MuX). The ml MSV-induced tumors MSV2 rapidly transforms cells of various species in culture regressed in the majority of birds. The present studies describe ' To whom requests for reprints should be addressed. the cell biology, virology, and pathology of ml MSV-induced 2 The abbreviations used are: MSV, Moloney murine sarcoma virus; ml MSV, quail tumors and describe some of the immune responses of the ml isolate of Moloney murine sarcoma virus; M-MuLV, Moloney murine tumor-bearing and regressor quail. leukemia virus; mos, Moloney murine sarcoma virus acquired sequences in the viral genome [supersedes the former usage of src (6)]; pP60OM, phosphorylated polyprotein with a molecular weight of 60,000 specified by the group antigen gene, gag; ASV, avian sarcoma virus; ppBO"0, phosphoprotein with a molecular MATERIALS AND METHODS weight of 60,000 specified by avian sarcoma virus transforming gene, src; MuX, murine xenotropic leukemia virus; B-MuX, BALB-derived murine xenotropic leu Animals. Embryonated Japanese quail eggs were obtained from Life kemia virus; mlMSV(B-MuX), ml isolate of Moloney murine sarcoma virus Sciences Inc., through the Office of Program Resources and Logistics, pseudotyped with BALB-derived murine xenotropic leukemia virus; SDS, sodium National Cancer Institute, and hatched at the Department of Poultry dodecyl sulfate; QEF, quail embryo fibroblast; PCS, fetal calf serum; FEF, feline embryo fibroblasts; S+L—, sarcoma positive, leukemia negative; FG10-IC, FG10 Science, University of Maryland. Newly hatched or adult quail were cells rescued with the 1C isolate of ecotropic Moloney murine leukemia virus; inoculated with virus or cells in a volume of 0.05 ml in the middorsal FeLV, feline leukemia virus; FIU, focus-inducing units; MuLV, murine leukemia area. Tumor development was monitored regularly, and palpable nod virus; MuLV-RM-Mmx. recombinant murine leukemia virus; FFU, focus-forming ules at the inoculation site of >3 mm were scored positive. Tumors units; gp70, glycoprotein with a molecular weight of 70,000; F-MuLV, Friend ranged in size from 3 to 24 mm. For transplantation, tumors were Eveline murine leukemia virus; p30, protein with a molecular weight of 30,000; pP60. phosphorylated polyprotein with a molecular weight of 60.000; cDNA. minced, and a cell volume of 0.5 ml was inoculated. Animals were complementary DNA; p28, protein with a molecular weight of 28,000; gPr85, sacrificed when moribund or at various stages of tumor growth. Extracts precursor glycoprotein with a molecular weight of 85,000; Pr65, precursor with of tumors were made at 10 and 20% (w/v) for virus and immunological a molecular weight of 65,000; p15E. protein with a molecular weight of 15,000 assays, respectively, the latter with 1% SDS. Sections of tissues and specified by envelope gene, env; ml MSV(MuLV), ml isolate of Moloney murine sarcoma virus pseudotyped with murine leukemia virus. tumors were stained with hematoxylin-eosin. Received December 7, 1981; accepted March 18, 1982. Cell Cultures. Primary QEF diploid cultures were prepared from JULY 1982 2523 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1982 American Association for Cancer Research. W. G. Robey et al. pools of 8-day-old eggs using trypsin (0.25%) and grown on McCoy's (34) and extracted with 4 ml of buffer containing 1% Triton X-100, Medium 5A with 10% PCS, 5% chicken serum, penicillin, and strep 0.5% sodium deoxycholate, 0.1% SDS, 0.01 M sodium phosphate (pH tomycin. After 7 days, secondary cultures were prepared in dishes with 7.5), 0.1 M NaCI, 1 HIM EDTA, and 200 units of Trasylol (Sigma) per ml the same medium at a cell density of 10"/sq cm. After DEAE-dextran (37). Cell extracts were clarified (20 min at 10,000 x g) and aliquoted treatment (25 ¿ig/ml, 1 ml/dish at 37°for 30 min), cells were washed for treatment with either goat or quail sera. Two immunoprecipitation and infected with 0.2 ml of various virus preparations. Twenty-four hr conditions were used because the Cowan I strain of Staphy/ococcus after infection, which was also 24 hr before inoculation, infected QEFs aureus (22) was not reactive with avian immunoglobulins (23) and the were put on McCoy's Medium 5A supplemented only with 5% normal formation of immune complexes by avian sera required higher salt quail serum. concentrations than did mammalian immune sera (18, 24). For goat Quail tumors were minced aseptically, and sections of these were antisera, cell extracts (1.0 ml) were incubated with 5 /il of normal goat also treated with trypsin and grown in McCoy's Medium 5A supple serum for 1 hr at 37°and then with 50 n\ of a 10% (v/v) suspension of mented with 10% PCS and 5% chicken serum. Many tumors grew formaldehyde-fixed S. aureus (22, 33). The mixtures were clarified (10 initially as mixtures of normal fibroblasts and transformed cells. Most min at 6000 x g) after incubation for 1 hr at 37°and then treated with tumor cultures could not be maintained for more than 2 to 3 months. In 5 /il of either antiserum against mIMSV pP60 or antiserum against 2 tumor cultures, loosely adherent transformed cells predominated, F-MuLV gp70. After incubation for 16 hr at 4°, immune complexes and these could be passed by shaking. After 1 year in culture, these were precipitated by the addition of 50 /il of a 10% suspension of S. were grown on McCoy's Medium 5A supplemented with 10% FCS. aureus and washed 3 times in cell extraction buffer. Immunoprecipi- Other cell lines used were diploid FEF, cat S+L- clone 81 cells, tated polypeptides were extracted from S. aureus by treatment with mouse S + L- clone FG10 cells, FG10-IC cells, B-MuX-infected human electrophoresis sample buffer for 3 min at 95°followed by centrifuga- embryonic lung cells, and normal mink lung cells (CCL-64). Cultures of tion (10 min at 6000 x g). In order to promote the formation of immune these were maintained in McCoy's Medium 5A with 5 to 15% FCS as complexes by quail sera, the NaCI and EDTA concentrations of cell detailed elsewhere (2,11,12,14).

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