VIROLOGY
70, 260-264
(1976)
Tumorigenicity and the Quantity of Virus DNA in PolyomaTransformed and Revertant Cell Lines LEO J. GRADY Division
of Laboratories
AND ARLENE
BELORIT
and Research, New York State Department
NORTH
of Health,
Albany,
New York 12201
Accepted December 22, 1975
The amount of virus DNA per cell was compared with the tumorigenicity of a polyoma-transformed line of mouse cells and two revertant lines selected for low saturation density after treatment with concanavalin A. The amount of virus DNA in cells of a tumor produced by inoculating the parent transformed line into syngeneic mice was also determined. Analysis of the reassociation kinetics suggests that all of these cells contain one copy of 35% of the virus genome per diploid quantity of cell DNA.
Several selection procedures have been reported (l-7) for the isolation from transformed cells of cell lines that have reverted in one or more of their growth properties to the nontransformed phenotype. In those revertants so far examined (4, 8), the number of copies of the virus genome per diploid quantity of cell DNA has been indistinguishable from that in the parent transformed cells. These results have led to the conclusion that reversion of various in vitro growth properties cannot be attributed to a shift in the ratio of virus to cell DNA. The possibility that the amount of virus DNA per diploid quantity of cell DNA might be related to the tumorigenicity of a cell line has also been investigated. Siegel and Levine (9) have reported no significant difference between two clones of SV40-transformed hamster heart cells with high and low oncogenic potential. Likewise, Gallimore et al. (10) found no correlation between the amount of adenovirus sequences in different lines of transformed rat cells and their tumorigenicity. We have now made similar observations with regard to cells transformed by polyoma virus by showing that the number of copies of the virus genome per diploid quantity of cell DNA is the same in a polyoma-transformed line of mouse cells that produces tumors with high efficiency in syngeneic mice and in two revertants of
the transformed cells that have low saturation densities in culture and reduced tumorigenicity in vivo. In addition, we have made the heretofore unreported observation that the ratio of virus to cell DNA found in transformed cells in vitro is unchanged in the cells of tumors that develop after injection of the transformed cells into syngeneic mice. PY AL/N, clone 3, a polyoma-transformed derivative of the AL/N line of mouse cells (11) was used in these experiments. Variants of PY AL/N cells that have low saturation densities in culture were selected by the method of Culp and Black (3). The general methods of cell culture have been published (12). Growth curves for two independently isolated revertant lines, designated A, and Ad, and for the parent PY AL/N cells are presented in Fig. 1. Although the revertants have the same growth rate as the PY AL/N cells, they have a markedly lower saturation density, about 5 x lo4 cells/cm”, which is similar to that of the nontransformed AL/N line (12). This low saturation density has been retained through 16 passages in culture. As shown in Table 1, the two revertant lines have more chromosomes per cell than the parent transformed cells. This phenomenon is often associated with reversion of the transformed phenotype (4, 8). 260
Copyright 0 1976by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT COMMUNICATIONS
1osO-
160 HOURS
FIG. 1. Growth curves of polyoma-transformed cells and the concanavalin A-selected revertants. Several clones with flat morphologies were chosen and recloned in Microtest II plates. Six clones which retained flat morphology and were positive for polyoma T-antigen (T-antigen tests were kindly carried out by Dr. K. K. Takemoto at the National Institutes of Health) were isolated. Two of these, designated A, and A,, were selected for further study. Growth rates and saturation densities were determined as previously described (I,!?). O-O, PY revertant A,. AL/N; A-A, revertant A,; A-A,
Measurements of the amount of DNA per cell revealed that while the revertants contain a little more DNA than the PY AL/N cells, both parent and revertant lines have about twice as much DNA per cell as diploid mouse embryo cells. Thus, the measured amount of DNA per cell agrees well with what would be expected from the data on the number of chromosomes per cell. With regard to tumorigenicity, it is clear from Table 1 that while the oncogenie potential of the revertants is significantly reduced compared to PY AL/N cells, they have not lost the ability to produce tumors. In contrast, the nontransformed AL/N cells, which have a saturation density similar to that of the revertants, did not produce tumors even with inocula as high as 5 x lo6 cells (12). These data emphasize that reversion of one or more features of the transformed pheno-
261
type in culture do not necessarily imply that the tumor-forming ability of the cells has been lost. The amount of virus DNA contained in the DNA of the various cells was determined on the basis of reassociation rate measurements as described by Gelb et al. (13). The kinetic analysis used the equations developed by Sharp et al. (14). In the case where no virus sequences are present in the cell DNA, the reassociation behavior of the [3Hlpolyoma DNA is represented by Eq. 1 of Sharp et al. (14). The lower, dotted line in Fig. 2 illustrates the expected reassociation rate under these circumstances. Note that the data obtained with mouse embryo and calf thymus DNA fall along this line. On this basis it is concluded that the 13Hlpolyoma DNA did not incorporate detectable levels of mouse DNA during growth of the virus (15). This is an important result upon which the validity of all of the other measurements rests. The two main features which emerged from the measurements made with DNA from the experimental cells were: (1) the ratio of virus to cell DNA was the same in all of the cell lines and also in cells from PY AL/N-induced tumors; and (2) no more than 50% of the virus genome was present per diploid quantity of cell DNA in any single cell. The latter situation can arise if the complete virus genome is represented randomly but no more than 0.5 copy per diploid cell, or if only a particular portion of the virus genome is retained in the cells of the various lines. Since the PY AL/N cells and both revertants had been cloned, it seemed unlikely that the experimental observation could be explained in terms of random representation of the whole virus genome . This conclusion was strengthened when Eq. 3 of Sharp et al. (14) was used to obtain a plot of the reassociation kinetics expected if the complete virus genome was present at 0.5 copy per diploid cell (Fig. 2, dashed line). There is little question that the actual data deviate significantly from this line. The second possibility, that the cells carry only a limited portion of the virus genome, was explored using Eq. 4 of Sharp
262
SHORT COMMUNICATIONS TABLE 1
NUMBER
OF CHROMOSOMES
Cell line I____-PY AL/N Rev. A, Rev. A, Mouse embryo
PER CELL,
Modal chromosome number”
75 100 100 40
AMOUNT
OF DNA PER CELL AND THE TUMORICENICITY STUDIED
DNA per cell” (Pg)
8.4 9.6 ND” 4.8
OF THE CELLS
Tumor incidence” after injection of 5 x 10” Cells
10” Cells
5 x 10” Cells
10” Cells
515 717 515 ND
313 414 213 ND
8/8 214 013 ND
818 o/5 014 ND
___~ .__ 0 Chromosome spreads were prepared from each cell line, as well as from secondary cultures of mouse embryo cells, using published procedures 119). Approximately 30 metaphase spreads were counted in each instance. h The DNA content per cell was measured by the technique of Leyva and Kelley (20). ’ Number of tumors formed per number of animals inoculated. The method for determining the tumorigenicity of the cell lines has been described (12). Mice were checked for tumors twice weekly for 12 weeks. Once started, tumors were never observed to regress and ultimately led to the death of the animals. (’ Not determined.
et al. (14). By making various assumptions regarding the fraction of the virus genome present and the frequency at which different segments occur, several curves can be generated that agree with the experimental results. However, the simplest solution consistent with the data is that there is a single copy of 35% of the virus genome per diploid quantity of cell DNA. The solid line in Fig. 2 was calculated on this basis. This conclusion is also supported by preliminary measurements which indicate that 30-40% of the polyoma genome is expressed in PY AL/N cells (L. J. Grady and W. P. Campbell, unpublished data). This last result indicates that at least 30% of the virus genome is present in the cells and places definite constraints on the assumptions that can be made in generating a curve to fit the experimental data. The most interesting aspect of this work is that only one-third of the virus genome, equivalent to 1.0 x 10” daltons of DNA, appears to be present in the cells studied. In terms of current knowledge regarding the proportion of the virus genome required for cell transformation, it should be noted that Graham et al. (16) have shown that the smallest fragment of DNA from adenoviruses 2 and 5 that possesses transforming activity has a MW of 1.0 x 10”. In the case of SV40, the smallest fragment
isolated so far that retains transforming activity contains about 1.8 x 10” daltons of DNA (17). In keeping with these results, it is possible that the amount of polyoma DNA in the transformed cells used in our experiments approaches the minimum essential for transformation. To summarize, the ratio of virus DNA to cell DNA is similar in the highly oncogenie parent cell line and in the revertants which have reduced tumorigenicity. This observation agrees with those of Siegel and Levine (9) using hamster cells transformed by SV40 and those of Gallimore et al. (10) using adenovirus-transformed rat cells. Furthermore, we have shown that there is no difference in this ratio between PY AL/N cells grown in culture and the tumors which they produce after injection into syngeneic mice. These data are consistent with the idea that neither phenotypic revertants nor tumors arise from subclasses of transformed cells in which the determining factor is an altered ratio of virus to cell DNA. The least complex kinetic analysis suggests that the cells contain one copy of 35% of the virus genome per diploid cell. However, it must be noted that in a number of cell lines transformed by adenovirus (10, 14) and in one cell line transformed by SV40 (IS), some portions of the virus genome have been shown to occur with
SHORT
263
COMMUNICATIONS
FIG. 2. Reassociation kinetics of 1”Hlpolyoma DNA in the presence of DNA from transformed, tumor, revertant, and nontransformed cells. DNA was prepared from the various cell lines, from tumors, and from mouse embryos by the procedure of McCarthy and Hoyer (21 I. Calf thymus DNA was purchased from Worthington Biochemical Corp. To obtain labeled virus DNA, secondary cultures of weanling mouse kidney cells were grown in a modified Eagle’s medium (Gibco #71035), supplemented with 10% Gibco fetal bovine serum (FBS), and infected with plaque-purified, small plaque polyoma virus at a multiplicity of infection of 0.1 PFU/cell. At 24, 72, 120, and 168 hr postinfection (p.i.) fresh medium was added containing, respectively, 5, 10, 15, and 20 &i/ml of [$H]thymidine (sp act, 53 Ci/mmole, New England Nuclear Corp.). At 216 hr p.i., the cells were harvested and treated with receptor-destroying enzyme (22). The released viruses were purified by banding in a CsCl equilibrium density gradient. The lowest virus band, p = 1.32 grams/ml, was collected, the viruses were lysed, and polyoma DNA I was isolated by centrifugation to equilibrium in a C&l-ethidium bromide gradient (23). The specific activity of the purified DNA was 7.5 x lo;’ cpm/kg. For hybridization reactions, each cell DNA preparation was suspended in 1.5 mM NaCl-0.15 mM sodium citrate at a concentration of 600 pg/ml and sheared at 25,000 psi in a French pressure cell. The DNA was then concentrated to 5-6 mg/ml and dialyzed into 0.9 M phosphate buffer (PB, consist.ing of equimolar concentrations of NaHpPO, and Na,HPO,) containing 10 n&f EDTA. The final DNA concentration was adjusted to 4.0 mg/ml with the same dialysis. buffer and then sheared, [“Hlpolyoma DNA was added to 0.003 pglml. The reaction mixture was denatured in a boiling water bath for 5 min, layered with paraffin oil, and then incubated at 60” for the duration of the experiment. To determine the extent of nonspecific binding to the column (about 2%‘0),a 0.3-ml aliquot was removed immediately after denaturation, diluted into 5.0 ml of 0.06 M PB + 0.4% sodium lauryl sulfate at 60”, and passed over a column of hydroxylapatite at 60”. DNA reassociation was followed by similar treatment of aliquots removed at various times after the start of the 60” incubation. The procedure for analyzing samples on hydroxylapatite has been described 124). The lower dotted line is the rate expected when no virus sequences are associated with the cell DNA. The upper dashed line is the anticipated rate if the complete virus genome is randomly represented at an average frequency of 0.5 copy per diploid amount of cell DNA. The solid line was calculated assuming that one copy of 35% of the virus genome is present per diploid cell. In all cases, tll$’ = 50. The source of unlabeled DNA was: (a) O---O, PY AL/N; A---A, tumors; O--O, mouse embryo; O---O, a reconstruction experiment in which the equivalent of 10 copies of unlabeled virus DNA was added. (b) m---m, revertant A,: i>-- ---G, revertant A,; A--A, calf thymus.
frequency than others. Only additional work with specific fragments of polyoma DNA will reveal whether a similar phenomenon also occurs in the cells that have been described. greater
ACKNOWLEDGMENTS We thank Dr. Lorraine Flaherty for assistance with the mouse tumors. This work was supported in part by NIH Research Grant Number CA 13401-03, awarded by the National Cancer Institute, PHSVDHEW.
REFERENCES l. POLLACK, R. E., GREEN, H., and TODARO, G. J., Proc. Nat. Acud. Sci. USA 60, 126-133 (1968). 2. CULP, L. A., GRIMES, W. J., and BLACK, P. H.,d. Cell Biol. 50, 682-690 (1971). 3. CULP, L. A.. and BLACK, P. H., J. Viral 9, 611620 (1972). 4. SHANI, M., RABINOWITZ, Z., and SACHS. L., J. Viral. 10, 456-461 (19721. 5. OZANNE, B., J. Viral. 12. 79-89 (1973). 6. VOGEL, A., RISSER, R., and POLLACK, R., J. Celi. Physiol. 82, 181-188 (1973).
264
SHORT
COMMUNICATIONS
7. VOGEL, A., and POLLACK, R., J. Cell Physiol. 82, 189-198 (1973). 8. OZANNE, B., SHARP, P. A., and SAMBROOK, J., J. Viral. 12, SO-98 (1973). 9. SIEGEL, S. E., and LEVINE, A. S., J. Nat. Cancer Inst. 49, 1667-1673 (1972). 10. GALLIMORE, P. H., SHARP, P. A., and SAMBROOK, J., J. Mol. Biol. 89, 49-72 (1974). 11. TAKEMOTO, K. K., TING, R. C. Y., OZER, H. L., and FABISCH, P., J. Nut. Cancerlnst. 41, 14011409 (1968). 12. GRADY, L. J., and NORTH, A. B., Exp. Cell Res. 87, 120-126 (1974). 13. GELB, L. D., KOHNE, D. E., and MARTIN, M. A., J. Mol. Biol. 57. 129-145 (1971). 14. SHARP, P. A., PETTERSSON, U., and SAMBROOK, J., J. Mol. Biol. 86, 709-726 (1974). 15. LAVI, S., and WINOCOUR, E., Virology 56, 296299 (1974). 16. GRAHAM, F. L., ABRAHAMS, P. J., MULDER, C., HEIJNEKER, H., WARNAAR, S. O., DE VRIES, F. A. J., FIERS, W., and VAN DER EB, A. J.,
Cold Spring Harbor Symp. Quant. Biol. 39, 637-650 (1974).
17. ABRAHAMS, P. J., MULDER, C., VAN DE VOORDE, A., WARNAAR, S. O., and VAN DER EB, A. J., J. Viral. 16, 818-823 (1975). 18. BOTCHAN, M., OZANNE, B., SUGDEN, B., SHARP, P. A., and SAMBROOK, J., Proc. Nat. Acad. Sci. USA 71, 4183-4187 (1974). 19. MILLER, 0. J., MILLER, D. A., KOURI, R. E., ALLDERDICE, P. W., DEV, V. G., GREWAL, M. S., and HUTTON, J. J., Proc. Nat. Acad. Sci. USA 68, 1530-1533 (1971). 20. LEYVA, A., JR., and KELLEY, W. N., Anal. Biothem. 62, 173-179 (1974). 21. MCCARTHY, B. J., and HOYER, B. H., Proc.
Nat. Acad. Sci. USA 52, 915-922 (1964). 22. CRAWFORD, L. V., In “Fundamental Techniques in Virology” (K. Habel and N. P. Salzman, eds.), pp. 75-81. Academic Press, New York and London, 1969. 23. RADLOFF, R., BAUER, W., and VINOGRAD, J., Proc. Nat. Acad. Sci. USA 57, 1514-1521 (1967). 24. GRADY, L. J., and CAMPBELL, W. P., Cancer Res. 35, 1559-1562 (1975).
70, 260-264
(1976)
Tumorigenicity and the Quantity of Virus DNA in PolyomaTransformed and Revertant Cell Lines LEO J. GRADY Division
of Laboratories
AND ARLENE
BELORIT
and Research, New York State Department
NORTH
of Health,
Albany,
New York 12201
Accepted December 22, 1975
The amount of virus DNA per cell was compared with the tumorigenicity of a polyoma-transformed line of mouse cells and two revertant lines selected for low saturation density after treatment with concanavalin A. The amount of virus DNA in cells of a tumor produced by inoculating the parent transformed line into syngeneic mice was also determined. Analysis of the reassociation kinetics suggests that all of these cells contain one copy of 35% of the virus genome per diploid quantity of cell DNA.
Several selection procedures have been reported (l-7) for the isolation from transformed cells of cell lines that have reverted in one or more of their growth properties to the nontransformed phenotype. In those revertants so far examined (4, 8), the number of copies of the virus genome per diploid quantity of cell DNA has been indistinguishable from that in the parent transformed cells. These results have led to the conclusion that reversion of various in vitro growth properties cannot be attributed to a shift in the ratio of virus to cell DNA. The possibility that the amount of virus DNA per diploid quantity of cell DNA might be related to the tumorigenicity of a cell line has also been investigated. Siegel and Levine (9) have reported no significant difference between two clones of SV40-transformed hamster heart cells with high and low oncogenic potential. Likewise, Gallimore et al. (10) found no correlation between the amount of adenovirus sequences in different lines of transformed rat cells and their tumorigenicity. We have now made similar observations with regard to cells transformed by polyoma virus by showing that the number of copies of the virus genome per diploid quantity of cell DNA is the same in a polyoma-transformed line of mouse cells that produces tumors with high efficiency in syngeneic mice and in two revertants of
the transformed cells that have low saturation densities in culture and reduced tumorigenicity in vivo. In addition, we have made the heretofore unreported observation that the ratio of virus to cell DNA found in transformed cells in vitro is unchanged in the cells of tumors that develop after injection of the transformed cells into syngeneic mice. PY AL/N, clone 3, a polyoma-transformed derivative of the AL/N line of mouse cells (11) was used in these experiments. Variants of PY AL/N cells that have low saturation densities in culture were selected by the method of Culp and Black (3). The general methods of cell culture have been published (12). Growth curves for two independently isolated revertant lines, designated A, and Ad, and for the parent PY AL/N cells are presented in Fig. 1. Although the revertants have the same growth rate as the PY AL/N cells, they have a markedly lower saturation density, about 5 x lo4 cells/cm”, which is similar to that of the nontransformed AL/N line (12). This low saturation density has been retained through 16 passages in culture. As shown in Table 1, the two revertant lines have more chromosomes per cell than the parent transformed cells. This phenomenon is often associated with reversion of the transformed phenotype (4, 8). 260
Copyright 0 1976by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT COMMUNICATIONS
1osO-
160 HOURS
FIG. 1. Growth curves of polyoma-transformed cells and the concanavalin A-selected revertants. Several clones with flat morphologies were chosen and recloned in Microtest II plates. Six clones which retained flat morphology and were positive for polyoma T-antigen (T-antigen tests were kindly carried out by Dr. K. K. Takemoto at the National Institutes of Health) were isolated. Two of these, designated A, and A,, were selected for further study. Growth rates and saturation densities were determined as previously described (I,!?). O-O, PY revertant A,. AL/N; A-A, revertant A,; A-A,
Measurements of the amount of DNA per cell revealed that while the revertants contain a little more DNA than the PY AL/N cells, both parent and revertant lines have about twice as much DNA per cell as diploid mouse embryo cells. Thus, the measured amount of DNA per cell agrees well with what would be expected from the data on the number of chromosomes per cell. With regard to tumorigenicity, it is clear from Table 1 that while the oncogenie potential of the revertants is significantly reduced compared to PY AL/N cells, they have not lost the ability to produce tumors. In contrast, the nontransformed AL/N cells, which have a saturation density similar to that of the revertants, did not produce tumors even with inocula as high as 5 x lo6 cells (12). These data emphasize that reversion of one or more features of the transformed pheno-
261
type in culture do not necessarily imply that the tumor-forming ability of the cells has been lost. The amount of virus DNA contained in the DNA of the various cells was determined on the basis of reassociation rate measurements as described by Gelb et al. (13). The kinetic analysis used the equations developed by Sharp et al. (14). In the case where no virus sequences are present in the cell DNA, the reassociation behavior of the [3Hlpolyoma DNA is represented by Eq. 1 of Sharp et al. (14). The lower, dotted line in Fig. 2 illustrates the expected reassociation rate under these circumstances. Note that the data obtained with mouse embryo and calf thymus DNA fall along this line. On this basis it is concluded that the 13Hlpolyoma DNA did not incorporate detectable levels of mouse DNA during growth of the virus (15). This is an important result upon which the validity of all of the other measurements rests. The two main features which emerged from the measurements made with DNA from the experimental cells were: (1) the ratio of virus to cell DNA was the same in all of the cell lines and also in cells from PY AL/N-induced tumors; and (2) no more than 50% of the virus genome was present per diploid quantity of cell DNA in any single cell. The latter situation can arise if the complete virus genome is represented randomly but no more than 0.5 copy per diploid cell, or if only a particular portion of the virus genome is retained in the cells of the various lines. Since the PY AL/N cells and both revertants had been cloned, it seemed unlikely that the experimental observation could be explained in terms of random representation of the whole virus genome . This conclusion was strengthened when Eq. 3 of Sharp et al. (14) was used to obtain a plot of the reassociation kinetics expected if the complete virus genome was present at 0.5 copy per diploid cell (Fig. 2, dashed line). There is little question that the actual data deviate significantly from this line. The second possibility, that the cells carry only a limited portion of the virus genome, was explored using Eq. 4 of Sharp
262
SHORT COMMUNICATIONS TABLE 1
NUMBER
OF CHROMOSOMES
Cell line I____-PY AL/N Rev. A, Rev. A, Mouse embryo
PER CELL,
Modal chromosome number”
75 100 100 40
AMOUNT
OF DNA PER CELL AND THE TUMORICENICITY STUDIED
DNA per cell” (Pg)
8.4 9.6 ND” 4.8
OF THE CELLS
Tumor incidence” after injection of 5 x 10” Cells
10” Cells
5 x 10” Cells
10” Cells
515 717 515 ND
313 414 213 ND
8/8 214 013 ND
818 o/5 014 ND
___~ .__ 0 Chromosome spreads were prepared from each cell line, as well as from secondary cultures of mouse embryo cells, using published procedures 119). Approximately 30 metaphase spreads were counted in each instance. h The DNA content per cell was measured by the technique of Leyva and Kelley (20). ’ Number of tumors formed per number of animals inoculated. The method for determining the tumorigenicity of the cell lines has been described (12). Mice were checked for tumors twice weekly for 12 weeks. Once started, tumors were never observed to regress and ultimately led to the death of the animals. (’ Not determined.
et al. (14). By making various assumptions regarding the fraction of the virus genome present and the frequency at which different segments occur, several curves can be generated that agree with the experimental results. However, the simplest solution consistent with the data is that there is a single copy of 35% of the virus genome per diploid quantity of cell DNA. The solid line in Fig. 2 was calculated on this basis. This conclusion is also supported by preliminary measurements which indicate that 30-40% of the polyoma genome is expressed in PY AL/N cells (L. J. Grady and W. P. Campbell, unpublished data). This last result indicates that at least 30% of the virus genome is present in the cells and places definite constraints on the assumptions that can be made in generating a curve to fit the experimental data. The most interesting aspect of this work is that only one-third of the virus genome, equivalent to 1.0 x 10” daltons of DNA, appears to be present in the cells studied. In terms of current knowledge regarding the proportion of the virus genome required for cell transformation, it should be noted that Graham et al. (16) have shown that the smallest fragment of DNA from adenoviruses 2 and 5 that possesses transforming activity has a MW of 1.0 x 10”. In the case of SV40, the smallest fragment
isolated so far that retains transforming activity contains about 1.8 x 10” daltons of DNA (17). In keeping with these results, it is possible that the amount of polyoma DNA in the transformed cells used in our experiments approaches the minimum essential for transformation. To summarize, the ratio of virus DNA to cell DNA is similar in the highly oncogenie parent cell line and in the revertants which have reduced tumorigenicity. This observation agrees with those of Siegel and Levine (9) using hamster cells transformed by SV40 and those of Gallimore et al. (10) using adenovirus-transformed rat cells. Furthermore, we have shown that there is no difference in this ratio between PY AL/N cells grown in culture and the tumors which they produce after injection into syngeneic mice. These data are consistent with the idea that neither phenotypic revertants nor tumors arise from subclasses of transformed cells in which the determining factor is an altered ratio of virus to cell DNA. The least complex kinetic analysis suggests that the cells contain one copy of 35% of the virus genome per diploid cell. However, it must be noted that in a number of cell lines transformed by adenovirus (10, 14) and in one cell line transformed by SV40 (IS), some portions of the virus genome have been shown to occur with
SHORT
263
COMMUNICATIONS
FIG. 2. Reassociation kinetics of 1”Hlpolyoma DNA in the presence of DNA from transformed, tumor, revertant, and nontransformed cells. DNA was prepared from the various cell lines, from tumors, and from mouse embryos by the procedure of McCarthy and Hoyer (21 I. Calf thymus DNA was purchased from Worthington Biochemical Corp. To obtain labeled virus DNA, secondary cultures of weanling mouse kidney cells were grown in a modified Eagle’s medium (Gibco #71035), supplemented with 10% Gibco fetal bovine serum (FBS), and infected with plaque-purified, small plaque polyoma virus at a multiplicity of infection of 0.1 PFU/cell. At 24, 72, 120, and 168 hr postinfection (p.i.) fresh medium was added containing, respectively, 5, 10, 15, and 20 &i/ml of [$H]thymidine (sp act, 53 Ci/mmole, New England Nuclear Corp.). At 216 hr p.i., the cells were harvested and treated with receptor-destroying enzyme (22). The released viruses were purified by banding in a CsCl equilibrium density gradient. The lowest virus band, p = 1.32 grams/ml, was collected, the viruses were lysed, and polyoma DNA I was isolated by centrifugation to equilibrium in a C&l-ethidium bromide gradient (23). The specific activity of the purified DNA was 7.5 x lo;’ cpm/kg. For hybridization reactions, each cell DNA preparation was suspended in 1.5 mM NaCl-0.15 mM sodium citrate at a concentration of 600 pg/ml and sheared at 25,000 psi in a French pressure cell. The DNA was then concentrated to 5-6 mg/ml and dialyzed into 0.9 M phosphate buffer (PB, consist.ing of equimolar concentrations of NaHpPO, and Na,HPO,) containing 10 n&f EDTA. The final DNA concentration was adjusted to 4.0 mg/ml with the same dialysis. buffer and then sheared, [“Hlpolyoma DNA was added to 0.003 pglml. The reaction mixture was denatured in a boiling water bath for 5 min, layered with paraffin oil, and then incubated at 60” for the duration of the experiment. To determine the extent of nonspecific binding to the column (about 2%‘0),a 0.3-ml aliquot was removed immediately after denaturation, diluted into 5.0 ml of 0.06 M PB + 0.4% sodium lauryl sulfate at 60”, and passed over a column of hydroxylapatite at 60”. DNA reassociation was followed by similar treatment of aliquots removed at various times after the start of the 60” incubation. The procedure for analyzing samples on hydroxylapatite has been described 124). The lower dotted line is the rate expected when no virus sequences are associated with the cell DNA. The upper dashed line is the anticipated rate if the complete virus genome is randomly represented at an average frequency of 0.5 copy per diploid amount of cell DNA. The solid line was calculated assuming that one copy of 35% of the virus genome is present per diploid cell. In all cases, tll$’ = 50. The source of unlabeled DNA was: (a) O---O, PY AL/N; A---A, tumors; O--O, mouse embryo; O---O, a reconstruction experiment in which the equivalent of 10 copies of unlabeled virus DNA was added. (b) m---m, revertant A,: i>-- ---G, revertant A,; A--A, calf thymus.
frequency than others. Only additional work with specific fragments of polyoma DNA will reveal whether a similar phenomenon also occurs in the cells that have been described. greater
ACKNOWLEDGMENTS We thank Dr. Lorraine Flaherty for assistance with the mouse tumors. This work was supported in part by NIH Research Grant Number CA 13401-03, awarded by the National Cancer Institute, PHSVDHEW.
REFERENCES l. POLLACK, R. E., GREEN, H., and TODARO, G. J., Proc. Nat. Acud. Sci. USA 60, 126-133 (1968). 2. CULP, L. A., GRIMES, W. J., and BLACK, P. H.,d. Cell Biol. 50, 682-690 (1971). 3. CULP, L. A.. and BLACK, P. H., J. Viral 9, 611620 (1972). 4. SHANI, M., RABINOWITZ, Z., and SACHS. L., J. Viral. 10, 456-461 (19721. 5. OZANNE, B., J. Viral. 12. 79-89 (1973). 6. VOGEL, A., RISSER, R., and POLLACK, R., J. Celi. Physiol. 82, 181-188 (1973).
264
SHORT
COMMUNICATIONS
7. VOGEL, A., and POLLACK, R., J. Cell Physiol. 82, 189-198 (1973). 8. OZANNE, B., SHARP, P. A., and SAMBROOK, J., J. Viral. 12, SO-98 (1973). 9. SIEGEL, S. E., and LEVINE, A. S., J. Nat. Cancer Inst. 49, 1667-1673 (1972). 10. GALLIMORE, P. H., SHARP, P. A., and SAMBROOK, J., J. Mol. Biol. 89, 49-72 (1974). 11. TAKEMOTO, K. K., TING, R. C. Y., OZER, H. L., and FABISCH, P., J. Nut. Cancerlnst. 41, 14011409 (1968). 12. GRADY, L. J., and NORTH, A. B., Exp. Cell Res. 87, 120-126 (1974). 13. GELB, L. D., KOHNE, D. E., and MARTIN, M. A., J. Mol. Biol. 57. 129-145 (1971). 14. SHARP, P. A., PETTERSSON, U., and SAMBROOK, J., J. Mol. Biol. 86, 709-726 (1974). 15. LAVI, S., and WINOCOUR, E., Virology 56, 296299 (1974). 16. GRAHAM, F. L., ABRAHAMS, P. J., MULDER, C., HEIJNEKER, H., WARNAAR, S. O., DE VRIES, F. A. J., FIERS, W., and VAN DER EB, A. J.,
Cold Spring Harbor Symp. Quant. Biol. 39, 637-650 (1974).
17. ABRAHAMS, P. J., MULDER, C., VAN DE VOORDE, A., WARNAAR, S. O., and VAN DER EB, A. J., J. Viral. 16, 818-823 (1975). 18. BOTCHAN, M., OZANNE, B., SUGDEN, B., SHARP, P. A., and SAMBROOK, J., Proc. Nat. Acad. Sci. USA 71, 4183-4187 (1974). 19. MILLER, 0. J., MILLER, D. A., KOURI, R. E., ALLDERDICE, P. W., DEV, V. G., GREWAL, M. S., and HUTTON, J. J., Proc. Nat. Acad. Sci. USA 68, 1530-1533 (1971). 20. LEYVA, A., JR., and KELLEY, W. N., Anal. Biothem. 62, 173-179 (1974). 21. MCCARTHY, B. J., and HOYER, B. H., Proc.
Nat. Acad. Sci. USA 52, 915-922 (1964). 22. CRAWFORD, L. V., In “Fundamental Techniques in Virology” (K. Habel and N. P. Salzman, eds.), pp. 75-81. Academic Press, New York and London, 1969. 23. RADLOFF, R., BAUER, W., and VINOGRAD, J., Proc. Nat. Acad. Sci. USA 57, 1514-1521 (1967). 24. GRADY, L. J., and CAMPBELL, W. P., Cancer Res. 35, 1559-1562 (1975).
