Transformation of mammalian cells by avian myelocytomatosis virus and avian erythroblastosis virus

VIROLOGY 98, 461-466 (1979)

Transformation

of Mammalian Cells by Avian Myelocytomatosis and Avian Erythroblastosis Virus

Virus

KRISTINA QUADE Departmmt

of Twnour Virology, Imperial Cancer Research Fund, Lincoln’s Inn Fields, ~~~~

WC%4 ?%PX,~ngla~

Accepted Jduly IS, 1979 Clones of rat cells transformed by avian myelocytomatosis virus strain MC29 and avian e~~obla~o~s virus have beenisolated. These cells display an altered mo~holo~ and are able to form colonies in soft agar. Focus-forming virus can be rescued from the transformed rat cells by fusion with chick cells.

Avian retrovi~ses can be divided into three groups: sarcoma viruses (ASV), nondefective lymphatic leukemia viruses (NDLV), and defective acute leukemia viruses (1, 2). Defective leukemia viruses (DLV) include avian myelocytomatosis virus strain MC29 and avian erythroblastosis virus (AEV). Both MC29 and AEV are probably defective in all three genes necessary for replication, namely gag, pal, and env (8, 4). Furthermore, they may contain one genes which are unrelated to the ASV src gene (5-7). MC29 and AEV are capable of transforming hematopoietic cells both in viva and in vitro. The normal target cell for transformation by MC29 is an immature myeloid cell and for AEV an immature erythroid cell (8); however, these viruses are also able to replicate in other hematopoietic cell types which they are unable to transform (T. Graf, personal communication). MC29 and AEV can also transform avian fibroblasts (9-12). In contrast ASV, which efficiently transform avian fibroblasts, are unable to transform and replicate only poorly, if at all, in cells of hematopoietic origin (2). ASV can however transform fibroblasts and certain other cells from heterologous species, including both rodents and primates (13). In view of the different transforming capabilities of- the src and putative one genes, it was of interest to determine if MC29 and AEV are also capable of infecting and transforming cellsof heterologoushosts.

MC29, with the subgroup D transfo~ation-defective (td) Carr-Zilber associated virus (CZAV) as helper virus and AEV strain ES4, with CZAV or td B77 (subgroup C) as helper viruses, were the gift of Dr. T. Graf. Rous associated virus-l (RAV-l), CZAV (used in rescue experiments), and ASV strain B’77 were kindly provided by Dr. J. Wyke. Brown leghorn x white leghorn C/E chick embryo fibroblasts (CEF) were obtained from Wickham Laboratories, Wickham, Hampshire, U. K., and primary cultures were prepared by standard procedures (14). DLV-infected CEF were grown in Ham’s FlO medium (Flow Laboratories) supplemented with 10%tryptose phosphate broth (TP), 5% calf serum, 2% chick serum, and 0.5% dimethylsulfoxide (DMSO). Uninfected and NDLVinfected CEF were grown in Dulbecco’s modified Eagle’s medium (DME) supplemented with 10% TP, 5% calf serum, and 1% chick serum. Infections of CEF were performed in similar media but with the omission of DMSO and addition of 2 pg/ml Polybrene. Focus assays were performed essentially as described by Graf (15). Line F2408 Fischer rat cells (16) were obtained from Dr. A. M. Fried. A variant resistant to 10 pg/ml t~ogua~ne was isolated from the rat cells after mutagenesis with ethylmethanesulfonate(Sigma)and a clone(208F) was obtained by micromanipulation. Rat cells were grown in DME supplemented with 10% fetal calf serum.

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0042-6822’79/140461-05$02.00/O Copyright All rights

0 1979 by Academic Press, Inc. of reproduction in any form reserved.

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Two methods were used to obtain DLVtransformed rat cells. CEF were infected with MC29 or AEV at a multiplicity of infection of 0.1-0.3 focus-forming units (FFU) per cell and passaged two to three times. Supernatant was harvested at 2hr intervals, clarified by centrifugation, and used to infect 208F monolayers at an equivalent CEF multiplicity of l-10 FFUl cell. Alternatively, 208F cells were cocultivated with mitomyein C killed transformed CEF essentially by the method of Chen et al. (17). After 14-21 days areas of putative transformation were aspirated with a capillary pipet and transferred to new dishes. Clones were obtained either by micromanipulation (18) or as colonies in agar suspension culture (19) seeded at low density (loo- 1000 cells/liO-mm dish). The method of transformation and isolation for each cell clone is summarized in Table 1. Both methods have permitted the isolation of AEV-transformed cells, but MC29transformed cells have as yet been obtained only by coeultivation. In addition, for both

methods parallel cultures were mock infected or infected with the helper viruses alone; no transformed clones were obtained from these control cultures. The morphology of the 208F cells was altered by infection with MC29 or AEV (Fig. 1). MC29-transformed clones are more refractile and spindle shaped in appearance than uninfected 208F cells; the monolayer, however, remains relatively flat, even at confluence. Clone MCla in particular but also clone MC2 tend to shed cells into the medium. The AEV-transformed clones are more similar to B77-transformed rat cells in morphology; they are more refractile and rounded than MC29-transformed clones. At confluence clone ATla and AT2 cells become very rounded as do both B77transformed clones. In contrast, monolayers of AC3 and AC4a remain relatively flat at confluence, All the clones examined have an approximately diploid rat karyotype (40-42 chromosomes). Transformation was assayed by the increased ability of the cells to form colonies

TABLE 1 MC29 ANDAEV-TRANSFORMEDRATCELLLINES Virus rescued Clone

Virus”

MCla

MC29 (CZAV) MC29 (CZAV) MC29 (CZAV) AEV (td B77) AEV (tdB77) AEV (CZAV) AEV (CZAV) Uninfected B77 B77

MC2 MC3 ATla AT2 AC3 AC4a 208F Bl B2

Method of transformation Cocultivation

Method of isolatioi? M-+M

Colony formatioiY

CEF

+RAV-1

+tdB77

+CZAV

5.3

4.6

4.6

-

3.0

Cocultivation

M

13.8

4.0

2.6

-

-

Cocuttivation

M

8.0

3.3

3.7

4.0

-

14.1

0

4.0

3.8

2.0

Direct infection

M-+M

Cocultivation

M

23.8

4.8

-

-

-

Cocultivation

C

10.9

5.3

-

-

-

Cocultivation

c+c

40.3

3.9

3.5

3.0

-


0 5.3 6.3

0 -

0 -

0 -

~oeultivation Direct infection

M C M

11.2 20.3

a Virus pseudotype with which the rat cells were infected. B M = micromanipulation; C = colony in 0.33% agar; + = clone + subclone. c Percentage of cells giving rise ‘to colonies in 0.33% agar when seeded at 100-1000 cells/tiO-mm dish. d Virus titers (two to three passages after fusion), given as log,, FFUlml. CEF = fusion with uninfected CEF; +RAV-1, +tdB77, +CZAV = fusion with preinfected CEF; - = not tested.

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in soft agar (Table 1). Macroscopic colonies were counted after 14-21 days incubation at 37”. All clones showed a greatly increased ability to grow in agar. Colonies from AT2 and AC4a were similar to those produced by B’7’7-transformed cells; how-

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ever, colonies from ATla and AC3, and the MC29-transformed cells tended to be smaller and less variable in size. In order to provide evidence that the observed transformation was due to infection by MC29 and AEV, infectious virus was

FIG. 1. Phase-contrast micrographs of 208F cells and their MC29-, AEV-, and B’77-transformed derivatives (100x ).

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rescued by polyethylene glycol (PEG)induced fusion of the rat cells with uninfected or NDLV-infected CEF. Fusions were performed by a modification of the procedures of Steimer and Boettiger (20). Briefly, 1 x lo6 mitomycin C killed rat cells were seeded with l-2 x lo6 CEF, either uninfected or preinfected with RAV-1, td B77, or CZAV (“complementation rescue assay”). After 16-24 hr they were fused with 50% PEG. The cells were passaged two to three times and the supernatants assayed for focus-forming virus. The results are summarized in Table 1. Virus was rescued from most clones without addition of exogenous helper virus. Clone ATla, however, was consistently negative (in 5 experiments) for virus rescue with uninfected CEF; in addition, the supernatant from this clone contained no reverse transcriptase activity (unpublished data). Virus could be rescued from ATla (in 4 of 13 experiments) when fused with NDLV preinfected CEF. Virus rescued from MC29-transformed clones produced characteristic small foci of tightly packed refractile cells (11); virus from AEV-transformed clones produced diffuse foci of fusiform refractile cells (12). The ability to rescue focus-forming virus without additional helper virus may be explained by noting that DLV stocks typically contain helper virus in an excess of approximately 7- to loo-fold in MC29 and 25- to lOOO-fold in AEV (5, 6, 21). Thus the rat cells themselves may contain the necessary helper virus to allow efficient rescue of the MC29 and AEV genomes. It has previously been shown that when rat cells are exposed to high titers of avian retrovirus, the majority contain rescuable virus genomes although only a small proportion are altered phenotypically (22). Neither MC29 nor AEV contain nucleic acid sequences related to the WC gene of ASV (5-7). It has been suggested that each contains instead unique sequences, putative one genes, which are responsible for their transforming ability. The recent isolation of an AEV mutant temperature-sensitive in its ability to block hemoglobin production in the host cell (28) indicates that a viral gene product is indeed necessary

for maintenance of a transformation-like function in erythroblasts. Whether the transformation of avian fibroblasts or the mammalian cells studied here result from the action of the same gene is as yet unknown; however, we have preliminary evidence (Hayman and Quade, unpublished data) which indicates that the characteristic gag-related proteins of 110,000 daltons in MC29 (21) and 75,000 daltons in AEV (24) are synthesized in these transformed rat clones. These clones should be useful in elucidating the transforming functions of these viruses. In addition they may prove valuable reagents for isolating antisera specific to the putative transforming proteins of these viruses. ACKNOWLEDGMENTS The author thanks Drs. C. Dickson, T. Graf, M. Hayman, G. Peters, and J. Wyke for helpful discussion and advice on the preparation of the manuscript. The author also thanks Ms. J. Newton for the typing of the manuscript. This work was performed during the tenure of a fellowship from the Leukemia Society of America. REFERENCES HANAFUSA, H., In “Comprehensive Virology’ (H. Fraenkel-Conrat and R. Wagner, eds.), Vol. 10, pp. 401-483. Plenum, New York, 19’77. 2. GRAF, T., and BEUG, H., Biochim. Bio&s. Acta. 516, 269-299 (19’78). 8. BISTER, K., and VOGT, P. K., Virology 88, 213-221 (1978). I. HAYMAN, M. J., ROYER-POKORA,B., and GRAF, T., Virology 92, 31-45 (1979). 5. DUESBERG,P. H., BISTER, K., and VOGT, P. K., Z’roc. Nut. Ad. Sci. USA 74,4320-4324 (1977). 6. ST~HELIN, D., and GRAF, T., Cell 13, 745-750 (1978). 7. SHEINESS, D., FANSHIER, L., and BISHOP,J. M., J. Viral. 28, 600-610 (1978). 8. GRAF, T., BEUG, H., ROYER-POKORA,B., and MEYER-GLAUNER, W., In “Differentiation of Normal and Neoplastic Hematopoietic Cells” (B. Clarkson, P. A. Marks, and J. E. Till, eds.), pp. 625-639. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., 1978. 9. LANGLOIS, A. J., SANKARAN, S., HSIUNG, P. L., ~~~BEARD, J. W.,J. Viral. 1,1082-1084@67). 10. ISH&~AKI, R., and SHIMIZU, T., Cancer Res. 36, 2827-2831 (1970). 11. GRAF, T., Virology 54, 398-413 (1973). 1.

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12. GRAF, T., ROYER-POKORA,B., SCHUBERT,G. E., 19. MACPHERSON,I., In “Tissue Culture Methods and and BEUG, H., Virology 71, 423-433 (19’76). Applications” (P. F. Kruse Jr. and M. K. Patterson Jr., eds.), pp. 276-280. Academic 13. TOOZE, J. (ed.), “The Molecular Biology of Tumour Viruses,” pp. 539-540. Cold Spring Press, New York, 1973. Harbor Laboratory, Cold Spring Harbor, 20. STEIMER, K. S., and BOETTIGER,D., J. Viral. 23, N. Y., 1973. 133-141 (1977). 14. VOGT, P. K., In “Fundamental Techniques in 21. BISTER, K., HAYMAN, M. J., and VOGT, P. K., Virology” (K. Habel and N. P. Salzman, eds.), Virology 82, 431-448 (1977). pp. 198-211. Academic Press, New York, 1969. 22. BOETTIGER, D., Cell 3, ‘71-76 (1974). 15. GRAF, T., Virology 50, 567-578 (1972). 23. GRAF, T., ADE, N., and BEUG, H., Nature 16. MISHRA, N. K., and RYAN, W. L., Znt. J. Cancer (London) 275,496-501 (1978). 11, 123-130 (1973). 17. CHEN, Y. C., HAYMAN, M. J., and VOGT, P. K., 24. HAYMAN, M. J., BISTER, K., VOGT, P. K., ROYERCell 11, 513-521 (1977). POKORA, B., and GRAF, T., In “Avian RNA 18. MACPHERSON,I., In “Fundamental Techniques in Tumor Viruses” (S. Barlati and C. de GiuliVirology” (K. Habel and N. P. Salzman, eds.), Morghen, eds.), pp. 214-226, Piccin Editore, pp. 17-20. Academic Press, New York, 1969. Padua, Italy, 1978.