- Email: [email protected]
Transformation of guinea pig embryo cells by a murine sarcoma virus
VIROLOGY
48,
841-843
~~~~~forrn~~ion
(1972)
of Guinea
Pig Embryo
Mouse sarcoma virus (MSV) is capable of transforming mouse, rat, hamster, bovine and human cells & vitro (1-7). The K&ten mouse sarcoma virus (Ki-MSV) (8), a MSV isolate from a rat-passaged murine erythoblastosis virus, shares not, only the groupspecific (gs) complement-fixing (CF) antigen of the murine leukemia-sarcoma virus group (9) but also many in v&o properties with the Maloney (10) and Harvey (11) strains of MSV. The present communication reports results of experiments showing that guinea pig embryo (GPE) cells can be transformed morphologically in vitro by IG-MSV, and that’ these morphologically transformed cells contain both infectious virus and gs antigen. Ki-MSV stock [supernatant fluids from a IQ-MSV-transformed rat cell line Ko. 58967 (7)] was kindly supplied by Dr. V. Klement,, Childrens Hospital, Los Angeles, CA and passed once in our laboratory in NI!I Swiss mouse embryo (NIH-ME) (pool 5630) or Fisher rat embryo (RE) (pool 5632) cultures. Its infectivity titer was 3.6 X lo4 -ME and 1.9 X 10” FFU/ ml in Fisher RE cells, respectively. Guinea pig embryo cells were prepared from theNIH strain (obtained from the animal production of National Institutes of Health). Cells were grown and maintained in Eagle’s minimum essential medium with 10 % fetal bovine serum and 2 mM glutamine, 100 pg penicillin, and 100 mg streptomycin/ml (EMEM + 10 % FIB). A suspension of freshly trypsinized primary GPE cells was centrifuged, resuspendedin fresh media, seededon BO-mm Falcon plastic dishes (2 X lo5 cells per plate) and incubated at 37” under 5 % GOa in air. After 24 hr the cultures were fed with fresh media and infected with either virus stock (pool 5638 or pool 5632) and returned to the incubator. Control cultures consisted
Cells of the samepreparation of cells in EM!QM + IO 70 FBS but were not inoculated with virus. Both inoculated and control cultures were fed with fresh media 24 hr later and they were thereafter refed at 3-. to 4-day intervals. Three weeks after inoculat,ion, some cultures were subdivided by trypsin treatment and continued as serial cell lines. Approximately 15-20 days after infection, foci consisting of fusiform cells mixed with round cells began to appear in the infected cultures. These foci increased in size during the next week. After one transfer, quit’e disr;inct foci were seen in the infecte cultures, but none was observed in the uninfected controls (Fig. IA. and B). The transformed foci were similar to those obtained with Ki-MS’C’ in mouse and rat embryo cells, and they contained many cytoplasmic vacuoles. The foci gradually increased in size and showed a pronounced proliferative effect with multilayered cell growth (Fig. 16). Characteristics of t,wo lines of K-?&W transformed cells are shown in Table P. The transformed cells were found to release virus cont.inuously. Cell-free preparations of supernatant *did from the in vitro transformed cultums produced similar altered foci in NIBand GPE cells. It should be noted here that in some experiments foci in GPE cells not readily visible upon inoculation I ever, after one or t\vo transfers distinct foci were seenin the infected culture. This may due to low titers of the virus preparation. The transformed cells contained high titers of gs antigen characteristic of the murine sarcoma-leukemia virus complex (8; 1,2). Sucrose gradient banding of the supernatant fluid showed a peak of radioactivity at a buoyant density of approximately 1.16 g;:‘lml: the density characteristic of murine sarcoma-leukemia virus complex (13). Studies are in progress to determine whether the
842
SHORT
COMMUNICATIONS
Since the guinea pig cell-grown virus transformed GPE cells readily (9-13 days after inoculation) whereas the original virus did not do it readily, the virus may have acquired new properties (Table 1). Focal areas of transformed cells were isolated by cloning. After four transfers, morphologically transformed cells could still be seen. These cultures still contained high titers of gs antigen. The susceptibility of NIH GPE cells to other C-type sarcoma viruses were carried out. Sarcoma viruses tested were the Moloney, Harvey isolates of MSV, and Theilen strain of feline sarcoma virus. No transformation has yet been observed with these viruses in GPE cells. The present results show that Ki-MSV infects and transforms GPE cells and indicate that members of the murine sarcoma-leukemia virus complex, particularly Ki-MSV, exhibit a broader host range than was hithert,o believed. ACKNOWLEDGMENTS We thank Dr. Robert J. Huebner for his encouragement and valuable comments and Mr. H. C. Turner for supervising complement-fixation tests. This work was conducted under Contract PH 43-70-20688 within Special Viral Cancer Program of the National Cancer Institute. REFERENCES
FIG. 1. Cultures of guinea pig embryo cells: (X 70). A. Uninfected control, second passage. B. A transformed focus appearing on GPE cells at 21 days after infection, second passage. C. A more advanced focus seen on GPE cells at 28 days after infection, second passage.
transformed cells produce tumors when inoculated into homologous hosts and also to further characterize the transformed cells.
1. HARTLEY, J. W., and ROWE, W. P., Proc. AYat. Acad. Sci. U.S.A. 55, 780-786 (1966). 1. TING, R. C., Virology 28, 783-785 (1966). 3. SIMONS, P. C., BASSIN, R. H., and HARVEY, J. J., Proc. Sot. Exp. Biol. Med. 125, 12421246 (1967). .J. THOMAS, M., BOIRON, M., STOYTCHKOV, Y., and LASNERET, J., Virology 36, 514-518 (1968). 5. BOIRON, M., BERNARD, C., and CHUAT, J. C,. Proc. Amer. Ass. Cuncer Rss. 10, 8 (1969). 6. AARONSON, S. A., and TODARO, G. J., Nature (London) 225, 458-459 (1970). 7. KLEMENT, V., FREEDMAN, M. H., MCALLISTER, R. M., NELSON-REES, W. A., and HUEBNER, R. J., J. Nat. Cancer Inst. 47, 65-73 (1971). 8. KIRSTEN, W. H., I nd MAYER, L. A., J. Nat. Cancer Inst. 39, 311335 (1967). 9. HARTLEY, J. W., ROWE, W. P., C~PPS, W. I., and HUEBNER, R.. J., J. Vi’irol. 3, 126-132 (1969).
SHORT
COMMUNICATIONS TABLE
CHAXACTERISTICS
OF KI-MSV-TRANSFORMED
Cell lines
Subculture level
R-804.4-i
Primary 2 4 6 Primary 4 6
B-804A-2
L1Reciprocal b Not tested. t Days after
of CF
titer.
PIG
EMBRYO (GPE)
CEIL LIXES
CummuExamination of culture sqernatant fractions .- -.~.-lathe no. CF titer” vs ~of days MSV rat Presence of Induction of foci after ixoculation in cells after inserum BH-nridine fection labeled virus NIIX-ME Fisher-RE NIH-GPE 21 26 39 50 21 39 50 0 indicates
0 >4 16 32 4 32 >32 a titer
NT6 NT NT yes NT NT yes
XT + (6P + (6) ST + (61 + (7) NT
NT + 03) i- (13) NT + (13) +@.?I NT
NT - (13) -+ (a) ST + (9) T (10) XT
<1:2.
infection.
10. MOLOXEY, J. B., Nat. Cancer Inst. Monogr. 22, 139 (1966). 12. HARVEY, J. J., Nature (Lo&on) 204, 1104 (1964). II. HARTLEY, .J. W., ROWE, W. P., CAPPS, W. I., and HIJUNE~., R. J., Proc. Nat. Acad. Sci. U.S.A. 53, 931-938 (1965). 13. SOMERS, K., and KIT, S., Virology 46, 774-
785 (1971) ~
1 GUINEA
J. s, KI-IfX c.
E‘. I)EMOT,WX
ii‘. G. DWH El:. Y cm Department
of Virus
Microbiological Bethesda, Accepted
Research
Associatesp Marylund March
20014 8, 1.978
Inc.
48,
841-843
~~~~~forrn~~ion
(1972)
of Guinea
Pig Embryo
Mouse sarcoma virus (MSV) is capable of transforming mouse, rat, hamster, bovine and human cells & vitro (1-7). The K&ten mouse sarcoma virus (Ki-MSV) (8), a MSV isolate from a rat-passaged murine erythoblastosis virus, shares not, only the groupspecific (gs) complement-fixing (CF) antigen of the murine leukemia-sarcoma virus group (9) but also many in v&o properties with the Maloney (10) and Harvey (11) strains of MSV. The present communication reports results of experiments showing that guinea pig embryo (GPE) cells can be transformed morphologically in vitro by IG-MSV, and that’ these morphologically transformed cells contain both infectious virus and gs antigen. Ki-MSV stock [supernatant fluids from a IQ-MSV-transformed rat cell line Ko. 58967 (7)] was kindly supplied by Dr. V. Klement,, Childrens Hospital, Los Angeles, CA and passed once in our laboratory in NI!I Swiss mouse embryo (NIH-ME) (pool 5630) or Fisher rat embryo (RE) (pool 5632) cultures. Its infectivity titer was 3.6 X lo4 -ME and 1.9 X 10” FFU/ ml in Fisher RE cells, respectively. Guinea pig embryo cells were prepared from theNIH strain (obtained from the animal production of National Institutes of Health). Cells were grown and maintained in Eagle’s minimum essential medium with 10 % fetal bovine serum and 2 mM glutamine, 100 pg penicillin, and 100 mg streptomycin/ml (EMEM + 10 % FIB). A suspension of freshly trypsinized primary GPE cells was centrifuged, resuspendedin fresh media, seededon BO-mm Falcon plastic dishes (2 X lo5 cells per plate) and incubated at 37” under 5 % GOa in air. After 24 hr the cultures were fed with fresh media and infected with either virus stock (pool 5638 or pool 5632) and returned to the incubator. Control cultures consisted
Cells of the samepreparation of cells in EM!QM + IO 70 FBS but were not inoculated with virus. Both inoculated and control cultures were fed with fresh media 24 hr later and they were thereafter refed at 3-. to 4-day intervals. Three weeks after inoculat,ion, some cultures were subdivided by trypsin treatment and continued as serial cell lines. Approximately 15-20 days after infection, foci consisting of fusiform cells mixed with round cells began to appear in the infected cultures. These foci increased in size during the next week. After one transfer, quit’e disr;inct foci were seen in the infecte cultures, but none was observed in the uninfected controls (Fig. IA. and B). The transformed foci were similar to those obtained with Ki-MS’C’ in mouse and rat embryo cells, and they contained many cytoplasmic vacuoles. The foci gradually increased in size and showed a pronounced proliferative effect with multilayered cell growth (Fig. 16). Characteristics of t,wo lines of K-?&W transformed cells are shown in Table P. The transformed cells were found to release virus cont.inuously. Cell-free preparations of supernatant *did from the in vitro transformed cultums produced similar altered foci in NIBand GPE cells. It should be noted here that in some experiments foci in GPE cells not readily visible upon inoculation I ever, after one or t\vo transfers distinct foci were seenin the infected culture. This may due to low titers of the virus preparation. The transformed cells contained high titers of gs antigen characteristic of the murine sarcoma-leukemia virus complex (8; 1,2). Sucrose gradient banding of the supernatant fluid showed a peak of radioactivity at a buoyant density of approximately 1.16 g;:‘lml: the density characteristic of murine sarcoma-leukemia virus complex (13). Studies are in progress to determine whether the
842
SHORT
COMMUNICATIONS
Since the guinea pig cell-grown virus transformed GPE cells readily (9-13 days after inoculation) whereas the original virus did not do it readily, the virus may have acquired new properties (Table 1). Focal areas of transformed cells were isolated by cloning. After four transfers, morphologically transformed cells could still be seen. These cultures still contained high titers of gs antigen. The susceptibility of NIH GPE cells to other C-type sarcoma viruses were carried out. Sarcoma viruses tested were the Moloney, Harvey isolates of MSV, and Theilen strain of feline sarcoma virus. No transformation has yet been observed with these viruses in GPE cells. The present results show that Ki-MSV infects and transforms GPE cells and indicate that members of the murine sarcoma-leukemia virus complex, particularly Ki-MSV, exhibit a broader host range than was hithert,o believed. ACKNOWLEDGMENTS We thank Dr. Robert J. Huebner for his encouragement and valuable comments and Mr. H. C. Turner for supervising complement-fixation tests. This work was conducted under Contract PH 43-70-20688 within Special Viral Cancer Program of the National Cancer Institute. REFERENCES
FIG. 1. Cultures of guinea pig embryo cells: (X 70). A. Uninfected control, second passage. B. A transformed focus appearing on GPE cells at 21 days after infection, second passage. C. A more advanced focus seen on GPE cells at 28 days after infection, second passage.
transformed cells produce tumors when inoculated into homologous hosts and also to further characterize the transformed cells.
1. HARTLEY, J. W., and ROWE, W. P., Proc. AYat. Acad. Sci. U.S.A. 55, 780-786 (1966). 1. TING, R. C., Virology 28, 783-785 (1966). 3. SIMONS, P. C., BASSIN, R. H., and HARVEY, J. J., Proc. Sot. Exp. Biol. Med. 125, 12421246 (1967). .J. THOMAS, M., BOIRON, M., STOYTCHKOV, Y., and LASNERET, J., Virology 36, 514-518 (1968). 5. BOIRON, M., BERNARD, C., and CHUAT, J. C,. Proc. Amer. Ass. Cuncer Rss. 10, 8 (1969). 6. AARONSON, S. A., and TODARO, G. J., Nature (London) 225, 458-459 (1970). 7. KLEMENT, V., FREEDMAN, M. H., MCALLISTER, R. M., NELSON-REES, W. A., and HUEBNER, R. J., J. Nat. Cancer Inst. 47, 65-73 (1971). 8. KIRSTEN, W. H., I nd MAYER, L. A., J. Nat. Cancer Inst. 39, 311335 (1967). 9. HARTLEY, J. W., ROWE, W. P., C~PPS, W. I., and HUEBNER, R.. J., J. Vi’irol. 3, 126-132 (1969).
SHORT
COMMUNICATIONS TABLE
CHAXACTERISTICS
OF KI-MSV-TRANSFORMED
Cell lines
Subculture level
R-804.4-i
Primary 2 4 6 Primary 4 6
B-804A-2
L1Reciprocal b Not tested. t Days after
of CF
titer.
PIG
EMBRYO (GPE)
CEIL LIXES
CummuExamination of culture sqernatant fractions .- -.~.-lathe no. CF titer” vs ~of days MSV rat Presence of Induction of foci after ixoculation in cells after inserum BH-nridine fection labeled virus NIIX-ME Fisher-RE NIH-GPE 21 26 39 50 21 39 50 0 indicates
0 >4 16 32 4 32 >32 a titer
NT6 NT NT yes NT NT yes
XT + (6P + (6) ST + (61 + (7) NT
NT + 03) i- (13) NT + (13) +@.?I NT
NT - (13) -+ (a) ST + (9) T (10) XT
<1:2.
infection.
10. MOLOXEY, J. B., Nat. Cancer Inst. Monogr. 22, 139 (1966). 12. HARVEY, J. J., Nature (Lo&on) 204, 1104 (1964). II. HARTLEY, .J. W., ROWE, W. P., CAPPS, W. I., and HIJUNE~., R. J., Proc. Nat. Acad. Sci. U.S.A. 53, 931-938 (1965). 13. SOMERS, K., and KIT, S., Virology 46, 774-
785 (1971) ~
1 GUINEA
J. s, KI-IfX c.
E‘. I)EMOT,WX
ii‘. G. DWH El:. Y cm Department
of Virus
Microbiological Bethesda, Accepted
Research
Associatesp Marylund March
20014 8, 1.978
Inc.