VIROLOGY
70, 223-226
Wt’6)
Transformation
of Hamster Embryo Cells by Defective Polyoma Virus DNA
AIM0 SALMI,’ JUTTA SEEHAFER, Department
of Biochemistry,
University
of Alberta,
Accepted November
AND
JOHN S. COLTER
Edmonton,
Alberta,
Canada
T6G 2H7
1, 1975
Two size classes of defective Py DNA, having estimated sedimentation coefficients of 31 and 41s in alkaline sucrose gradients, were isolated from preparations of Py virus after four serial, high multiplicity passages in mouse kidney cultures. The two DNA preparations, after recovery from alkaline sucrose gradients, were reannealed and used to “infect” secondary cultures of hamster embryo iibroblasts. Four to five weeks later, colonies of densely packed cells appeared in the cultures, and after six passages, four clones (two from each of the 31 and 41s Py DNA-treated cultures) were isolated. Cells from each clonal isolate, while retaining a fibroblastlike morphology, have acquired the capacity for continuous in vitro growth, have lost the property of contact inhibition of growth, and produce the Py-specific T-antigen. These observations suggest strongly that the cells have been transformed by the defective Py-DNAs. Serial passage of polyoma (Py) virus at high multiplicity of infection in permissive cells leads to the production of virions containing defective, covalently closed circular (ccc) DNA. It has been reported that the sizes of these defective DNA species may range from 50 to 102% of that, of the infectious, unit-length viral genome (1-3). Those that have been characterized have been found to contain viral DNA only, and appear to have been produced either by deletion of certain segments of the genome or by deletion of certain segments coupled with repetition (tandem or otherwise) of others (3-5). It is possible that other species of defective Py DNA may contain both viral and cellular species, as has been found in the case of DNA from SV40 produced in cells infected at a high multiplicity (6). Py virions containing defective DNA are, by definition, noninfectious. They are replicated only in cells coinfected with virions containing complete genomes. However, it is reasonable to suspect that at least some classes of defective virions may ’ Permanent address: Department of Virology, University of Turku, Turku, Finland. 223 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
be capable of transforming cells, since a limited number of viral functions are expressed in transformed cells. The fact that, with the exception of cells transformed with certain temperature sensitive mutants (7, 81, Py virus cannot be rescued from transformed cells by fusion with cells permissive for Py virus replication gives credence to this proposition. Direct experimental evidence is provided by this communication, in which we describe the transformation of hamster embryo fibroblasts by defective Py DNA. Inocula of wild-type (large plaque) Py virus were prepared by infecting 2-day-old mice, establishing cultures from their kidneys 8-10 days later, and harvesting the cultures when lysis was complete (9). Preparations of virus containing high levels of defective virions were produced, as described previously (1O), by passaging the virus at high input multiplicity (lo-100 PFU/cell) in primary mouse kidney cultures grown in Dulbecco’s modified Eagle’s medium (DME) containing 5% fetal calf serum (FCS). The virus produced during the fourth passage was labeled in its DNA by adding t’4Clthymidine (5 &i/ml, New England Nuclear Corp.) to the medium
224
SHORT
COMMUNICATIONS
from 1.5 hr p.i. to lysis, and was purified as outlined previously (10). DNA was released from purified 14C-labeled virions by incubation for 10 min at 20” in 1% SDS, 5% 2-mercaptoethanol, 0.3 N NaOH, after which the various species of DNA were resolved by velocity centrifugation in linear (1530%) alkaline sucrose density gradients. Fractions (8 drop) were collected from the bottoms of the gradients, and the positions of the peaks were determined by measuring the radioactivity in a 2-~1 aliquot of each fraction. An illustrative profile is shown in Fig. 1. Fulllength Py DNA, which sediments with an estimated sedimentation coefficient of 53 S, was only partially resolved in these preparations from at least one other size class of sedimentation coefficient about 50 S. However, two smaller size classes of cccDNA (here designated 41 and 31 S, corresponding to approximately 90% and 80% of the complete genome) were resolved clearly from one another and from the larger species. Fractions from three parallel gradients containing 41 and 31 S Py DNA were pooled separately. The denatured DNA in the pooled fractions was reannealed by a modification of
FRACTION
NUM8ER
FIG. 1. Velocity centrifugation in alkaline sucrose density gradient of [L4C]thymidine-labeled DNA isolated from Py virus after four serial, high multiplicity passages in mouse kidney cells. Centrifugation was for 195 min at 20” and 45,000 rpm (Beckman SW 50.1 rotor) in a 15-30% linear sucrose gradient containing 0.1% SDS, 0.001 M EDTA and 0.2 N NaOH. Sedimentation was from right to left. Fractions indicated by I-/ were pooled for further studies.
the procedure that has been used for reannealing denatured 4X-174 DNA (11). In brief, this involved adding NaCl to the pooled fractions to give a final concentration of 1 M, heating the samples for 10 min at 50”, adjusting the pH to 7.2 with 1 M NaH,PO,, and dialyzing against PBS (pH 7.2) containing 0.001 M EDTA. The extent of reannealing, estimated from sedimentation analyses in neutral sucrose density gradients (5-20% in 1 M NaCl, 0.01 M Tris, pH 7.9, 0.01 M EDTA; 3 hr at 20” and 40,000 rpm in a Beckman SW 50.1 rotor), was found to be 100 and 80% for the 31 and 41 S DNA, respectively. Reannealed 31 and 41 S Py DNA were examined for transforming capacity in secondary cultures of hamster embryo (HE) cells, and for infectivity (by plaque assay and virus production) in secondary mouse embryo fibroblast and mouse kidney cultures. At the same time, as a check on the methodology, the infectivity of 53 S Py DNA (isolated from virus preparations free of defective virions, and reannealed as described above after recovery from an alkaline sucrose gradient) was measured. In all cases, cells were “infected” with the DNA preparations according to the DEAEdextran method first described by McCutchan and Pagan0 (12). As expected, neither of the defective Py DNA preparations was found to be infectious, while the 53 S species was fully infectious. Cultures of HE cells (in 30-mm plastic petri dishes), which had been exposed to reannealed 31 and 41 S Py DNA as well as control HE cells, were maintained for 3 weeks in DME-10% FCS with weekly changes of medium. The cells were then collected by trypsinization, transferred to plastic tissue culture flasks (75 cm’?, and held for an additional 2 weeks in DME-5% FCS. At that stage, colonies of densely packed cells were visible in those cultures which had been exposed to both species of defective Py DNA. The gross appearance of representative cultures after staining with Giemsa stain is shown in Fig. 2, in which the colonies of cells are visible in the DNA-treated, but not the control cells. The treated cells were then passaged and grown to confluency six times in DME-5%
SHORT
COMMUNICATIONS
225
FCS, using a 15 split at each passage. times before being examined (on cover slip After the sixth passage the cells were cultures) for the presence of Py-virus Tplated at low cell density (103and 104cells! antigen by fluorescent antibody staining. 50-mm plastic petri dish for cells exposed Two rat antisera against Py T antigen to 41 and 31s Py DNA, respectively) for were used, one produced in this laboratory and the other kindly provided by Dr. cloning. Clones which grew up from each set of Michele Fluck, Department of Pathology, cultures were selected and recloned imme- Harvard Medical School; the FITC-anti rat diately. From the second cloning, two IgG was purchased from Miles Laboratoclonal isolates from each of the 41 and 31 S ries. All four lines were found to be posiPy DNA-treated cells were selected. Cells tive for Py T antigen with both antisera, of all four clonal isolates have retained the unstained cells being rare and randomly tibroblastic morphology of the HE cells distributed. Cells exhibiting the nuclear from which they were derived, but all have fluorescence typical of Py virus-translost the property of contact inhibition of formed cells are shown in Fig. 3. Included growth, and exhibit the phenomenon of in this study as a positive control was a line of Py virus-transformed cells produced “piling up” common to all virus-transin this laboratory. Control HE cells were formed cells. Cells isolated from cultures treated with 41s Py DNA grow rapidly in unstained, as were cells of our four clonal isolates after staining with either control BME-5% FCS, while those from cultures treated with 31s Py DNA grow slowly in rat serum + FITC-anti rat IgG or hamster this medium and have been maintained in antiserum to H-50 (a SVBO-transformed BME-5% tryptosephosphate broth-lo% cell) + FITC-anti hamster IgG (Grand Island Biological Co.). FCS. Cells of the four clonal isolates were Considered in toto, the observations grown to confluency and passaged three that cells isolated from HE cultures ex-
FIG. 2. Appearance of Giemsa stained cultures of: (A) Control hamster embryo cells; (B) hamster embryo cells exposed to reannealed 41 S Py DNA, (C) hamster embryo cells exposed to reannealed 31 S Py DNA. Photographs were taken 5 weeks after exposure of the cultures to the defective Py DNAs.
226
FIG.
defective
SHORT
3. Intranuclear fluorescent (41 S) Py DNA.
staining
COMMUNICATIONS
for Py-virus
posed to two different size classes of defective Py DNA have acquired the capacity for continuous in vitro- growth, have lost the property of contact inhibition of growth, and contain the Py T antigen, provide rather convincing evidence for the transforming capacity of these defective Py DNAs, and show that the entire viral genome is not required for transformation. They suggest further that many, perhaps most, Py virus-transformed cells have been transformed by defective viral-DNA species, which would in turn explain the failure to rescue infectious virus from such cells. ACKNOWLEDGMENTS The authors wish to express their thanks to Pat Carpenter and Irene Korolak for valuable technical assistance, and to Mr. Perry D’Obrenan and Cathy Hicks for their help in preparing the figures. The studies were supported by grants from the Medical Research Council of Canada (MT-1191) and the National Cancer Institute of Canada. Dr. A.
T-antigen
in hamster
embryo cells transformed
Salmi was the recipient of a University Killam Postdoctoral Fellowship.
by
of Alberta,
REFERENCES 1. BLACKSTEIN, M. E., STANNERS, C. P., and FARMILO, A. J., J. Mol. Biol. 42, 301-313 (1969). 2. FRIED, M., J. Virol. 13, 939-946 (1974). 3. GRIFFIN, B. E., and FRIED, M., Nature 256, 175179 (1975). 4. ROBBERSON, D. L., and FRIED, M., Proc. Nat. Acad. Sci. USA 71, 3497-3501 (1974). 5. FOLK, W. R., and WANG, H-C. E., Virology 61, 140-155 (1974). 6. LAVI, S., and WINOCOUR, E., J. Viral. 9, 309-316 (1972). 7. SUMMERS, J., and VOGT, M., In “Second Lepetit Symposium: The Biology of Oncogenic Viruses” (L. G. Silvestri, ed.) p. 306-311 (1970). North Holland Publishing Co., Amsterdam. 8. FOLK, W. R., J. Viral. 11, 424-431 (1973). 9. WINOCOUR, E., Virology 19, 158-168 (1963). 10. SEEHAFER, J., SALMI, A., SCRABA, D. G., and COLTER, J. S., Virology 66, 192-205 (1975). J., and 11. POUWELS, P. H., VAN ROTTERDAM, COHEN, J. A., J. Mol. Biol. 40, 379-390 (1969). 12. MCCUTCHAN, J. H., and PAGANO, J. S., J. Nat. Cancer Inst. 41, 351-357 (1968).
70, 223-226
Wt’6)
Transformation
of Hamster Embryo Cells by Defective Polyoma Virus DNA
AIM0 SALMI,’ JUTTA SEEHAFER, Department
of Biochemistry,
University
of Alberta,
Accepted November
AND
JOHN S. COLTER
Edmonton,
Alberta,
Canada
T6G 2H7
1, 1975
Two size classes of defective Py DNA, having estimated sedimentation coefficients of 31 and 41s in alkaline sucrose gradients, were isolated from preparations of Py virus after four serial, high multiplicity passages in mouse kidney cultures. The two DNA preparations, after recovery from alkaline sucrose gradients, were reannealed and used to “infect” secondary cultures of hamster embryo iibroblasts. Four to five weeks later, colonies of densely packed cells appeared in the cultures, and after six passages, four clones (two from each of the 31 and 41s Py DNA-treated cultures) were isolated. Cells from each clonal isolate, while retaining a fibroblastlike morphology, have acquired the capacity for continuous in vitro growth, have lost the property of contact inhibition of growth, and produce the Py-specific T-antigen. These observations suggest strongly that the cells have been transformed by the defective Py-DNAs. Serial passage of polyoma (Py) virus at high multiplicity of infection in permissive cells leads to the production of virions containing defective, covalently closed circular (ccc) DNA. It has been reported that the sizes of these defective DNA species may range from 50 to 102% of that, of the infectious, unit-length viral genome (1-3). Those that have been characterized have been found to contain viral DNA only, and appear to have been produced either by deletion of certain segments of the genome or by deletion of certain segments coupled with repetition (tandem or otherwise) of others (3-5). It is possible that other species of defective Py DNA may contain both viral and cellular species, as has been found in the case of DNA from SV40 produced in cells infected at a high multiplicity (6). Py virions containing defective DNA are, by definition, noninfectious. They are replicated only in cells coinfected with virions containing complete genomes. However, it is reasonable to suspect that at least some classes of defective virions may ’ Permanent address: Department of Virology, University of Turku, Turku, Finland. 223 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
be capable of transforming cells, since a limited number of viral functions are expressed in transformed cells. The fact that, with the exception of cells transformed with certain temperature sensitive mutants (7, 81, Py virus cannot be rescued from transformed cells by fusion with cells permissive for Py virus replication gives credence to this proposition. Direct experimental evidence is provided by this communication, in which we describe the transformation of hamster embryo fibroblasts by defective Py DNA. Inocula of wild-type (large plaque) Py virus were prepared by infecting 2-day-old mice, establishing cultures from their kidneys 8-10 days later, and harvesting the cultures when lysis was complete (9). Preparations of virus containing high levels of defective virions were produced, as described previously (1O), by passaging the virus at high input multiplicity (lo-100 PFU/cell) in primary mouse kidney cultures grown in Dulbecco’s modified Eagle’s medium (DME) containing 5% fetal calf serum (FCS). The virus produced during the fourth passage was labeled in its DNA by adding t’4Clthymidine (5 &i/ml, New England Nuclear Corp.) to the medium
224
SHORT
COMMUNICATIONS
from 1.5 hr p.i. to lysis, and was purified as outlined previously (10). DNA was released from purified 14C-labeled virions by incubation for 10 min at 20” in 1% SDS, 5% 2-mercaptoethanol, 0.3 N NaOH, after which the various species of DNA were resolved by velocity centrifugation in linear (1530%) alkaline sucrose density gradients. Fractions (8 drop) were collected from the bottoms of the gradients, and the positions of the peaks were determined by measuring the radioactivity in a 2-~1 aliquot of each fraction. An illustrative profile is shown in Fig. 1. Fulllength Py DNA, which sediments with an estimated sedimentation coefficient of 53 S, was only partially resolved in these preparations from at least one other size class of sedimentation coefficient about 50 S. However, two smaller size classes of cccDNA (here designated 41 and 31 S, corresponding to approximately 90% and 80% of the complete genome) were resolved clearly from one another and from the larger species. Fractions from three parallel gradients containing 41 and 31 S Py DNA were pooled separately. The denatured DNA in the pooled fractions was reannealed by a modification of
FRACTION
NUM8ER
FIG. 1. Velocity centrifugation in alkaline sucrose density gradient of [L4C]thymidine-labeled DNA isolated from Py virus after four serial, high multiplicity passages in mouse kidney cells. Centrifugation was for 195 min at 20” and 45,000 rpm (Beckman SW 50.1 rotor) in a 15-30% linear sucrose gradient containing 0.1% SDS, 0.001 M EDTA and 0.2 N NaOH. Sedimentation was from right to left. Fractions indicated by I-/ were pooled for further studies.
the procedure that has been used for reannealing denatured 4X-174 DNA (11). In brief, this involved adding NaCl to the pooled fractions to give a final concentration of 1 M, heating the samples for 10 min at 50”, adjusting the pH to 7.2 with 1 M NaH,PO,, and dialyzing against PBS (pH 7.2) containing 0.001 M EDTA. The extent of reannealing, estimated from sedimentation analyses in neutral sucrose density gradients (5-20% in 1 M NaCl, 0.01 M Tris, pH 7.9, 0.01 M EDTA; 3 hr at 20” and 40,000 rpm in a Beckman SW 50.1 rotor), was found to be 100 and 80% for the 31 and 41 S DNA, respectively. Reannealed 31 and 41 S Py DNA were examined for transforming capacity in secondary cultures of hamster embryo (HE) cells, and for infectivity (by plaque assay and virus production) in secondary mouse embryo fibroblast and mouse kidney cultures. At the same time, as a check on the methodology, the infectivity of 53 S Py DNA (isolated from virus preparations free of defective virions, and reannealed as described above after recovery from an alkaline sucrose gradient) was measured. In all cases, cells were “infected” with the DNA preparations according to the DEAEdextran method first described by McCutchan and Pagan0 (12). As expected, neither of the defective Py DNA preparations was found to be infectious, while the 53 S species was fully infectious. Cultures of HE cells (in 30-mm plastic petri dishes), which had been exposed to reannealed 31 and 41 S Py DNA as well as control HE cells, were maintained for 3 weeks in DME-10% FCS with weekly changes of medium. The cells were then collected by trypsinization, transferred to plastic tissue culture flasks (75 cm’?, and held for an additional 2 weeks in DME-5% FCS. At that stage, colonies of densely packed cells were visible in those cultures which had been exposed to both species of defective Py DNA. The gross appearance of representative cultures after staining with Giemsa stain is shown in Fig. 2, in which the colonies of cells are visible in the DNA-treated, but not the control cells. The treated cells were then passaged and grown to confluency six times in DME-5%
SHORT
COMMUNICATIONS
225
FCS, using a 15 split at each passage. times before being examined (on cover slip After the sixth passage the cells were cultures) for the presence of Py-virus Tplated at low cell density (103and 104cells! antigen by fluorescent antibody staining. 50-mm plastic petri dish for cells exposed Two rat antisera against Py T antigen to 41 and 31s Py DNA, respectively) for were used, one produced in this laboratory and the other kindly provided by Dr. cloning. Clones which grew up from each set of Michele Fluck, Department of Pathology, cultures were selected and recloned imme- Harvard Medical School; the FITC-anti rat diately. From the second cloning, two IgG was purchased from Miles Laboratoclonal isolates from each of the 41 and 31 S ries. All four lines were found to be posiPy DNA-treated cells were selected. Cells tive for Py T antigen with both antisera, of all four clonal isolates have retained the unstained cells being rare and randomly tibroblastic morphology of the HE cells distributed. Cells exhibiting the nuclear from which they were derived, but all have fluorescence typical of Py virus-translost the property of contact inhibition of formed cells are shown in Fig. 3. Included growth, and exhibit the phenomenon of in this study as a positive control was a line of Py virus-transformed cells produced “piling up” common to all virus-transin this laboratory. Control HE cells were formed cells. Cells isolated from cultures treated with 41s Py DNA grow rapidly in unstained, as were cells of our four clonal isolates after staining with either control BME-5% FCS, while those from cultures treated with 31s Py DNA grow slowly in rat serum + FITC-anti rat IgG or hamster this medium and have been maintained in antiserum to H-50 (a SVBO-transformed BME-5% tryptosephosphate broth-lo% cell) + FITC-anti hamster IgG (Grand Island Biological Co.). FCS. Cells of the four clonal isolates were Considered in toto, the observations grown to confluency and passaged three that cells isolated from HE cultures ex-
FIG. 2. Appearance of Giemsa stained cultures of: (A) Control hamster embryo cells; (B) hamster embryo cells exposed to reannealed 41 S Py DNA, (C) hamster embryo cells exposed to reannealed 31 S Py DNA. Photographs were taken 5 weeks after exposure of the cultures to the defective Py DNAs.
226
FIG.
defective
SHORT
3. Intranuclear fluorescent (41 S) Py DNA.
staining
COMMUNICATIONS
for Py-virus
posed to two different size classes of defective Py DNA have acquired the capacity for continuous in vitro- growth, have lost the property of contact inhibition of growth, and contain the Py T antigen, provide rather convincing evidence for the transforming capacity of these defective Py DNAs, and show that the entire viral genome is not required for transformation. They suggest further that many, perhaps most, Py virus-transformed cells have been transformed by defective viral-DNA species, which would in turn explain the failure to rescue infectious virus from such cells. ACKNOWLEDGMENTS The authors wish to express their thanks to Pat Carpenter and Irene Korolak for valuable technical assistance, and to Mr. Perry D’Obrenan and Cathy Hicks for their help in preparing the figures. The studies were supported by grants from the Medical Research Council of Canada (MT-1191) and the National Cancer Institute of Canada. Dr. A.
T-antigen
in hamster
embryo cells transformed
Salmi was the recipient of a University Killam Postdoctoral Fellowship.
by
of Alberta,
REFERENCES 1. BLACKSTEIN, M. E., STANNERS, C. P., and FARMILO, A. J., J. Mol. Biol. 42, 301-313 (1969). 2. FRIED, M., J. Virol. 13, 939-946 (1974). 3. GRIFFIN, B. E., and FRIED, M., Nature 256, 175179 (1975). 4. ROBBERSON, D. L., and FRIED, M., Proc. Nat. Acad. Sci. USA 71, 3497-3501 (1974). 5. FOLK, W. R., and WANG, H-C. E., Virology 61, 140-155 (1974). 6. LAVI, S., and WINOCOUR, E., J. Viral. 9, 309-316 (1972). 7. SUMMERS, J., and VOGT, M., In “Second Lepetit Symposium: The Biology of Oncogenic Viruses” (L. G. Silvestri, ed.) p. 306-311 (1970). North Holland Publishing Co., Amsterdam. 8. FOLK, W. R., J. Viral. 11, 424-431 (1973). 9. WINOCOUR, E., Virology 19, 158-168 (1963). 10. SEEHAFER, J., SALMI, A., SCRABA, D. G., and COLTER, J. S., Virology 66, 192-205 (1975). J., and 11. POUWELS, P. H., VAN ROTTERDAM, COHEN, J. A., J. Mol. Biol. 40, 379-390 (1969). 12. MCCUTCHAN, J. H., and PAGANO, J. S., J. Nat. Cancer Inst. 41, 351-357 (1968).
