Brief Communication: Oncogenic Transformation of Murine Lymphoid Cells by

In Vitro Infection With Abelson Leukemia Virus 1,2

w. C. Raschke,3 P. Ralph,3 J. Watson,3 M. Sklar," and Helen Coon 5, 6

Inst54: 1249-1253, 1975. GENOMES OF C-TYPE RNA VIRUSES occur ubiquitously in mice; they are present in normal and tumor tissues (1). Several C-type RNA viruses have been isolated that cause leukemias and lymphomas when injected into normal recipients (2). The hematopoietic target cells for transformation appear to be virus specific. For example, Gross passage A, Moloney, Friend, and Rauscher leukemia viruses induce erythroleukemias or thymic lymphomas (3-6). The lack of in vitro systems for the transformation of hematopoietic cells by leukemia viruses has made this transformation difficult to study. In vivo, a latent period of many months between time of infection and appearance of neoplasms obscures the oncogenic process. Here we describe an in vitro system for the study of early events in the transformation of lymphoid cells by a C-type RNA virus. In this system, transformation depends on a particular virus and stimulation of lymphocytes. We used a C-type virus isolated by Abelson and Rabstein (7) from a Moloney leukemia virus-infected mouse pretreated with prednisolone from birth. The Abelson virus causes rapid induction of nonthymic lymphomas in mice (8, 9); the lymphomas appear to originate from bone marrowderived (B) lymphocytes (8). Virus replication and transformation requires DNA synthesis in the target cell (10). Although lymphocytes in culture normally do not divide, these cells can be stimulated to DNA synthesis and cell division by the addition of antigen or various mitogens such as bacterial lipopolysaccharide (LPS), which is specifically mitogenic for B lymphocytes (11). Since the target cells for Abelson virus seem to be B lymphocytes, spleen cultures were stimulated with bacterial LPS or with an tigen to ini tia te DNA syn thesis processes in B lymphocytes and then infected with Abelson virus. The infected spleen cells were injected into recipient mice to determine if oncogenic transformation had occurred. A number of tumors developed in these recipient mice. To distinguish whether the tumors were of donor rather than recipient origin, female

cells were infected in vitro and injected into male mice. The female karyotypes found in tumors arising in these male mice indicated that there was oncogenic transformation of cells infected in vitro. MATERIALS AND METHODS

Six-week-old iemale BALBjc mice were immunized with 0.2 ml of 10% sheep red blood cells (SRBC) injected iv to increase the number of lymphocytes specifically responding to SRBC. After 3 days, spleens were harvested and cultured in a modified MishellDutton system, as described in (11). Each I-ml culture contained 107 nucleated spleen cells plus either 3 X 106 SRBC, 5 fJg Salmonel'a typhosa LPS [a B-cel mitogen (J 1, 12); Difco Laboratories, Detroit, Mich.], or no mitogen or antigen. Appropriate cultures were infected with either Moloney virus or with an Abelson virus pool 3 days after being plated (see table 1). The Abelson virus pool was a 10% (wtjvol) extract of primary tumors clarified by centrifugation and filtered through a 0.45-fJ Millipore filter. Abelson virus preparations contain Moloney leukemia virus as well as the Abelson component (8). This pool had a tumorigenic dose of 103.6jml in newborn BALBjc mice and a Moloney virus titer of 106. 6 plaque-forming units (PFU)/ml, as determined by the XC plaque test with BALBjc embryo fibroblasts (13). The virus pool also contained the lactate dehydrogenase virus, an unrelated virus not required for tumorigenesis by Abelson virus (Sklar M: U npublished data). The Moloney leukemia virus pool (University Laboratories, Highland Park, N.].) was a 10% (wt/vol) extract of a leukemic BALBjc spleen reclarified and filtered through a 0.45-fJ Millipore filter. Its PFU titer was adjusted to 10 6.6jml by 1: 2 dilution of the filtrate. Since the Abelson component may be replication defective like the murine sarcoma viruses, in one experimental group the Abelson virus pool was diluted into the Moloney virus pool to keep Received October 23, 1974; accepted January 24, 1975. Supported by grants AI05875 and AI00430 (to Dr. Melvin Cohn) and All 1092 (to J.W.) from the National Institute of Allergy and Infectious Diseases; grant GB 37869 (to P.R.) from the National Science Foundation; and Jane Coffin Childs Postdoctoral Fellowship 61-345 (to W.R.'. 3 The Salk Institute for Biological Studies, P.O. Box 1809, San Diego, Calif. 92112. 4 National Institute of Allergy and Infectious Diseases, National Institutes of Health, Public Health Service, U.S. Department of Health, Education, and Welfare, Bethesda, Md. 20014. Present address: Department of Radiology, Stanford University Hospital, Stanford, Calif. 94305. 5 National Cancer Institute, National Institutes of Health. 6 We gratefully acknowledge the support of Drs. Melvin Cohn, Wallace Rowe, and Michael Potter; and the assistance with the karyology provided by Dr. Hayden Coon. 1

2

JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 54, NO.5, MAY 1975

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SUMMARY-Spleen cell cultures stimulated to DNA synthesis by antigen or mitogen were infected with Abelson virus, a C-type RNA virus inducing nonthymic lymphomas in mice. After 3 days the cells were transferred to mice and caused 100% incidence of lymphomas in as few as 29 days. That a number of the tumors were of donor origin, as shown by female karyotypes in recipient male mice, indicated that cells infected by virus in vitro were transformed. The process depended upon both virus and stimulation of lymphocytes in culture. Lymphoid tumors did not develop in mice receiving cells from virus-infected cultures not exposed to antigen or mitogen.-J Natl Cancer

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RASCHKE, RALPH, WATSON, SKLAR, AND COON

RESULTS

Table 1 shows that lymphoma developed in every mouse receiving cells treated in vitro with Abelson virus plus antigen or mitogen, with the exception of 6 nude mice that died during the observation period with no evidence of tumor at autopsy. Being athymic, TABLE

Mouse group

1.-Induction of lymphosarcomas by infection of spleen cultures in vitro with Abelson virus

Virus (ml)a Abelson

nude mice are immunologically deficient and therefore more susceptible to environmental infection. The tumors appeared as early as 29 days, and all tumors in these groups (4-8) occurred by 104 days after inoculation. Mice were moribund within 7 days of tumor detection. All tumors appeared in lymph nodes, including the parathymic nodes of the athymic nude mice, and occasionally spleen. Many tumorbearing animals also had abundant ascites associated with the pristane injection. All 28 tumors were readily transplantable. None of the mice with tumors had anti-SRBC activity or myeloma proteins in the serum. The predominant cell type in most tumors resembled a lymphoblast similar to that reported for other Abelson tumors (8, 9). The cells had deeply indented nuclei and no cytoplasmic granules. The few tumors exhibiting no blast cells had the appearance of medium-to-Iarge lymphocytes. No tumors developed in the recipients of control cultures lacking virus (group 1). Only one recipient of Moloney virus-infected cultures (group 2) developed a tumor, and this occurred 227 days after inoculation. Only 2 of 4 recipients of the Abelson virus-infected cultures lacking antigen or mitogen stimulation (group 3) developed tumors, after 163 and 266 days. The tumors in these 3 recipients were distinguished from all other tumors by their long latent period and their inability to be successfully transplanted. Furthermore, these 3 mice had much ascitic fluid but did not exhibit any other typical evidence of tumor, such as enlarged lymph nodes or spleen. These results indicated that the tumors in the Abelson groups did not result from the Moloney virus component. Stimulation of DNA synthesis with mitogen or appropriate antigen was necessary for tumorigenesis by spleen cells infected with Abelson virus in vitro.

Moloney

Antigen or mitogen b

Sex of recipient

N umber of mice with tumors/No. inoculated C BALB/c

1 2 3 4 5 6 7 8

91

0.1 0.1 0.1 0.01 0.01 0.01 0.01 0.2 0.1 O. 1 (ip) O. 1 (ip)

0.1

0.09 O. 09

SRBC SRBC None SRBC SRBC SRBC SRBC SRBC SRBC SRBC

F M M

LPS

F

F F F F

M F

M

M M

0/3 1/3 2/4 3/3 3/3 3/3

-

3/3

3/3

Day(s) killed

Tumors karyotyped

Tumors with female karyotypes

Nude

2/3 d 1/3' 1/3 e 2/3 d 3/3

227 163,266 29,45,45 38,48 45,48,56 35 63,91,104 84 72,91,91 50, 50 56,60,60 36,45,60

RAW 14

RAW 14

RAW 4

RAW 4

RAW 24 RAW 23, 26, 27

RAW 23

Virus added on 3d day of culture. Both Abelson and Moloney virus stocks had XC titers of 106.6 PFUjml. 3XI06 SRBC or 5 "g LPS (S. typhosa) added to I-ml cultures containing 107 spleen cells from female BALBjc mice primed 3 days previously with 0.2 ml of 10% SRBC iv. c After 6 days, cells were washed, pooled, and inject.ed ip into BALB/c or nude mice, 3-7X10 6 viable cells/3 cultures/mouse. d 1 nude mouse found dead; no tumors detected at autopsy. • 2 nude mice found dead; no tumors detected at autopsy. f Control group: Mice inoculated ip with 0.1 ml of same pool of Abelson virus used in vitro. a

b

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helper virus titer constant while reducing the titer of the Abelson component (table 1, group 6). Three days after infection of spleen cultures with virus, the cells were injected ip into BALBjc or nude mice (table 1). The congenitally athymic nude mice had been backcrossed into the BALB/c background for four generations (11). Cultures were injected into both male and female mice. Inoculation of BALB/c mice with pristane (a light mineral oil) before injection of tumor cells has greatly enhanced transplantation of plasmacytomas (14) and Abelson lymphomas without affecting the rapidity or total incidence of lymphoma induction by Abelson virus (15). Therefore, all mice were given ip injections of 0.5 ml pristane 1-3 weeks before cell injection. As a control for tumor induction by virus alone, 3 pristane-treated mice of each strain were inoculated ip with 0.1 mlof the same pool of Abelson virus used in vitro. Tumors that developed were classified by gross and microscopic pathology, and were tested for the presence of myeloma protein in the serum or ascitic fluid by agar electrophoresis and for production of antibody to SRBC by hemagglutination. Tumors in male mice were transplanted two to four times in male (recipient karyotype) mice. Mice were inoculated with colchicine, 1 J.Lg/g body weight. After 1.5 hours, cells were obtained from ascites and karyotyped by standard methods, with Giemsa and quinacrine mustard staining (16, 17).

TRANSFORMATION OF LYMPHOID CELLS IN VITRO

DISCUSSION

The finding that Abelson leukemia virus causes rapid induction of myelomas and lymphocytic neoplasms (8, 15) suggested to us that the virus might be used for oncogenic transformation in vitro. Productive infection of fibroblasts by murine leukemia viruses and transformation by murine sarcoma viruses had been studied extensively, but similar work with hematopoietic cells, the natural target of leukemia viruses, has not been reported. Our experiments show that it is possible to infect spleen cultures with Abelson leukemia virus and obtain lymphomas upon subsequent transfer of the cells to mice. Some of the tumors are derived from the cells cultured with virus, as shown by female donor karyotypes among tumor cells developing in male hosts. Male karyotypes also present in some of these tumors may represent normal invading host cells or secondary transformation of host cells by virus released from the transplanted infected cells. Because the tumors are carried by transp]antation in male mice, the background of male cells would be expected to increase with each passage; this would be due to recruitment of new host tumor cells transformed by the virus produced by the transplanted cells. The continued presence of female cells after four transplantations is compelling evidence that tumors arose from the female cells infected in vitro. We have subsequently confirmed these findings using a much easier to score chromosome translocation marker (18). Since these experiments have included an in vivo phase because spleen cells are not easily maintained in culture for long periods, it is difficult to determine if oncogenic even ts occurred in donor cells after

passage into mice. However, the success in tumor induction clearly depends on stimulation of the B lymphocytes in vitro as well as on inoculation with virus. Infected spleen cultures incubated with SRBC or LPS, in conditions leading to increased DNA synthesis and maturation of B lymphocytes to plasmacytes, produced lymphomas in all mice inoculated, whereas mice given infected but unstimulated spleen cells did not develop lymphomas. With suitable tissue culture conditions, it may be possible to demonstrate transformation of lymphoid cells entirely in vitro, as would be shown by unlimited growth in culture, presence of viral properties, and rapid appearance of lymphomas upon transplantation to mice. The neoplasms induced in vivo by Abelson leukemia virus appear to be of B-Iymphocyte origin (8). Tumors arising from the in vitro infection discussed here also exhibit properties of the B-Iymphocyte series. We showed that an Abelson lymphoma tumor (from group 4, table 1) adapted to culture is very sensitive to growth inhibition by LPS, a property not shown by thymus-derived lymphoma, myeloma, mastocytoma, or erythroleukemia (19). A study of a series of Abelson cell lines based on the inhibitory effects of three B-Iymphocyte mitogens (LPS, dextran sulfate, and tuberculin purified protein derivative) indicates that they represent B lymphocytes at various stages of differentiation from stem cell to early and late forms (20). With long-term (more than 30 days) pristane priming of recipients, Abelson virus also induces tumors of plasma cells, the terminal differentiation state ofB lymphocytes (15). In contrast to the lymphoblast tumors we produced, we have now obtained transformed plasmocytes by in vitro passaging of infected spleen cells in mice that had received longterm pristane treatment (Raschke W: Manuscript in preparation). Antigen-specific stimulation of DNA synthesis and separation of antigen-specific cells from the general population by various methods would allow the production of lymphomas and myelomas synthesizing antibody to any preselected antigen. Thus these experiments, as well as being a toolfor the study of transformation of lymphocytes by leukemia viruses in vitro, suggest model systems for study of cloned cell lines of antigen-sensitive cells, induction of immune responses, properties of B lymphocytes during differentiation, and the directed production of homogeneous antibody with known antigenbinding activity. REFERENCES (1) TODARO GJ, HUEBNER RJ: The viral,oncogene hypothesis: New evidence. Proc Natl Acad SCI USA 69:1009-1015, 1972 (2) RICH MA, SIEGLER R: Virus leukemia in the mouse. Annu Rev Microbiol21 :529-572, 1967 (3) GROSS L: "Spontaneous" leukemia developing in C3H mice following inoculation, in infancy, with AKleukemic extracts, or AK-embryos. Proc Soc Exp BioI Med 76 :27-32, 1951 (4) MOLONEY JB: Biological studies on a lymphoid-.1e~kemia virus extracted from Sarcoma 37, 1. OrIgm and introductory investigations. J Nat! Cancer lnst 24:933951, 1960

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The shortest latent periods were obtained for cultures infected with undiluted and tenfold-diluted Abelson virus. Contrary to expectation, by doubling the amount of Abelson virus added (group 7) or diluting the virus tenfold into Moloney virus (group 6), we obtained markedly longer latent periods. The latent periods for appearance of tumor showed no marked differences between nude and BALB/c mice nor between sexes in sensitivity to direct virus infection or transplantation. Of the seven tumors arising from transplants of in vitro-infected female cells into male mice, six were karyotyped (RAW 4, 14, 23, 24, 26, and 27). These represent four experimental groups (table 1). Four of these tumors were euploid (RAW 4,14,23, and 27); three (4, 14, and 23) contained cells definitely demonstrating the female karyotype. A karyotype of a metaphase from RAW 14 and one from 23 are shown in figure 1. No female cells were found in 13 metaphases from the sample of RAW 27. RAW 24 contained chromosome numbers ranging from 39 to 42, with a strong mode at 41. All RAW 24 meta phases analyzed had a Y chromosome and were therefore male. RAW 26 had 39 chromosomes, and though no Y chromosome was observed, no preparations were adequate for the unequivocal identification of two X chromosomes. Thus at least three and possibly four of six tumors contained cells with the female karyotype.

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(14)

(15) (16) (17)

(18)

(19) (20)

leukemia viruses in tissue culture. Proc Natl Acad Sci USA 63 :753-758, 1969 POTTER M, PUMPHREY JG, WALTERS JL: Growth of primary plasmacytomas in the mineral oil-conditioned peritoneal environment. J Nat! Cancer Inst 49 :305-308 1972 ' POTTER M, SKLAR MD, ROWE WP: Rapid viral induction of plasmacytomas in pristane-primed BALB/c mice. Science 182 :592-594, 1973 CASPERSSON T, ZECH L, MODEST EJ: Fluorescent labeling of chromosomal DNA: Superiority of quinacrine mustard to quinacrine. Science 170:762, 1970 MILLER OJ, MILLER DA, KOURI RE, et al: Identification of the mouse karyotype by quinacrine fluorescence and tentative assignment of seven linkage groups. Proc Natl Acad Sci USA 68:1530-1533, 1971 SKLAR MD, WHITE BJ, ROWE WP: Initiation of oncogenic transformation of mouse lymphocytes in vitro by Abelson leukemia virus. Proc Natl Acad Sci USA 71: 4077-4081, 1974 RALPH P, NAKOINZ I: Lipopolysaccharides inhibit lymphosarcoma cells of bone marrow origin. Nature (Lond) 249:49-51, 1974 RALPH P, NAKOINZ I, RASCHKE WC: Lymphosarcoma growth is selectively inhibited by B lymphocyte mitogens: LPS, dextran sulfate and PPD. Biochem Biophys Res Commun 61 :1268-1275, 1974

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(5) FRIEND C: Cell-free transmission in adult Swiss mice of a disease having the character of a leukemia. J Exp Med 105:307-318, 1957 (6) RAUSCHER FJ: A virus-induced disease of mice characterized by erythrocytopoieis and lymphoid leukemia. J Nat! Cancer Inst 29:515-543, 1962 (7) ABELSON HT, RABSTEIN LS: Influence of prednisolone on Moloney leukemogenic virus in BALB/c mice. Cancer Res 30:2208-2212, 1970 (8) - - - : Lymphosarcoma: Virus-induced thymic-independent disease in mice. Cancer Res 30:2213-2219, 1970. (9) SIEGLER R, ZAJDEL S, LANE I: Pathogenesis of Abelsonvirus-induced murine leukemia. J Nat! Cancer Inst 48:189-218, 1972 (10) TEMIN HM: Studies on carcinogenesis by avian sarcoma viruses. V. Requirement for a new DNA synthesis and for cell division. J Cell Physiol 69 :53-63, 1967 (11) WATSON J, EpSTEIN R, NAKOINZ I, et al: The role of humoral factors in the initiation of in vitro primary immune responses. II. Effects of lymphocyte mitogens. J Immunol 110 :43-52, 1973 (12) WATSON J, TRENKNER E, COHN M: The use of bacterial lipopolysaccharides to show that two signals are required for the induction of antibody synthesis. J Exp Med 138:699-714, 1973 (13) KLEMENT V, ROWE W, HARTLEY J, et al: Mixed culture cytopathogenicity: A new test for growth of murine

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I.-Quinacrine mustard-banding karyotype of metaphase from tumors RAW 14(A) and RAW 23(B). Note paired X chromosomes and absence of a Y chromosome.

FIGURE

RASCHKE, RALPH, WATSON, SKLAR, AND COON

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