Properties of clonal lines of murine sarcoma virus transformed Balb3T3 cells

VIROLOGY

38,174-202

(1969)

Short Properties

of Clonal

Sarcoma

Virus Balb/3T3

Communications fusiform cells that tend to grow over the adjacent normal cells. After the first week the majority of the foci either progressed no further or regressed. A small fraction of the foci, however, continued to grow and by 2 weeks some had reached a considerably larger size (Fig. 1B). In some cases, rather than spreading laterally, the focus increased in cell number by forming multiple cell layers (Fig. 1C). After 3-4 weeks, transformed colonies containing well over 1000 cells could be seen at the site of an initial focus (Fig. 1D). The factors that determine whether an acute focus will be self-limiting or progress to a transformed colony are not yet understood. The initial foci have been reported to form primarily by the spread of virus from an initially infected cell to adjacent cells, rather than by cell multiplication (7, 8). The RISV-transformed Balb/3T3 colonies, however, enlarge primarily by cell division. The increase in cell division in the cultures can be estimated by 3H-thymidine (3H-TdR) incorporation into cellular DNA in control and focus-containing Balb/3T3 monolayers. At 20 days after exposure to virus, plates that originally contained 400 foci incorporated 3H-TdR at over 50 times the rate of the uninfected controls (Fig. 2). Cultures with 50 initial foci that at 20 days had three large transformed colonies showed a fifteen-fold increase in the rate of DNA synthesis. Cells from areas of the Petri dish that contained transformed colonies were transferred at high dilution to new Petri dishes. The clones that grew up consisted of two morphological types; one, nor readily distinguishable from the untransformed Balb/3T3 cells and the second, composed of round or fusiform highly refractile cells that attached very poorly to the plastic substrate and tended to float off into the liquid medium. At low cell density the transformed clones generally grew more slowly than did the

Lines of Murine Transformed Cells

The Balb/3T3 cell line, derived from Balb/c mouse enbryo cultures, is a permanent line of contact-inhibited, non-tumorigenie cells (1, 2). The cells can be transformed in tissue culture by the oncogenic DNA virus, SV40. The transformants, when inoculated into Balb/c mice, are capable of producing tumors (2). In the present communication we report the isolation and characterization of several cloned lines of Balb/3T3 cells transformed by murine sarcoma virus (WV). These morphologically transformed lines release large quantities of virus and produce tumors rapidly in weanling Balb/c mice. One particular Balb/3T3 clone was used in all experiments. This clone is susceptible to focus formation by the mouse sarcoma virus pseudotypes that are capable of growing in Balb/c cells (3). Since the efficiency of focus formation is greatest when logarithmically growing cells are infected (/t), confluent monolayers were trypsinized and plated at a 1: 4 dilution on the day prior to infection. In various experiments, cells were exposed either to tumor extracts of a Balb/c leukemia virus pseudotype (5) of RISV (supplied by Dr. J. Hartley) or, to the supernates from Balb/3T3 cultures transformed with this virus preparation. Both the tumor extract and the virus produced in tissue culture contained a lo- to loo-fold excess of mouse leukemia virus (RILV) as determined by comparing the end-point of focus-forming activity with the end-point dilution for detection of MLV by the COMUL test (6). Foci were first seen at 2-3 days after infection but were routinely counted on day 7. A typical focus seen at 7 days is shown in Fig. IA. Note the refractile rounded and 174

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normal-appearing clones. This was due in part to the tendency of transformed cells to detach and float off into the medium. How ever, if the transformants were obtained free from the untransformed cells by cloning they were capable of growing well from single

cells. Six such clones have been isolated and recloned. They reached considerably higher saturation densities than the normals (> 2 X lo5 cells/cm2 vs. 5 X lo4 cells/cm2). The RISV-transformed clones, when grown to mass culture, produced tumors in meanling

FIG. IA 1. Transformed foci induced in Balb/3T3 by NW. examples of foci seen at 2 weeks, 11. an RISV-transformed the site of an acllte focus. All unstained. X55. FIG.

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COMMUNICATIONS

A. Acute focus, colony 4 weeks

7 days after infection, B., after infection, developing

C. at

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Balb/c mice. Using 5 X lo5 cells inoculated subcutaneously, four out of five animals developed tumors within 3 weeks. With frozen and thawed cells two of five animals also developed tumors. Animals inoculated with untransformed cells, or supernates from the transformed cells were negative. Each of the six l\ISV-transformed clones

FIG.

released mouse sarcoma virus as determined by the focus-forming ability on Balb/3T3. Frozen and thawed supernates that were filtered through i\Iillipore membranes with an average pore diameter of 0.45 P were used. The focus-forming activity in the fluids obtained 24 hours after medium change of confluent cultures depended on the particu-

1B

SHORT

COhDIU?u’ICATIONS

lar clone and ranged from 6.0 X lo* to 2.4 X lo5 focus-forming units (FFU) per milliliter. Some clones, once isolated, gave rise to homogeneous populations of round, refractile cells as described above. However, at least one clone (MSV-1) “spontaneously” yielded colonies with the typical Balb/3T3 morphology. The supernates from one of the

FIG.

177

clones that remained stable (clone 3) and clone 1 were tested for focus-forming activity at 30, 60, and 100 cell generations after transformation (Table 1). Clone 3 released 2 X lo4 to 4 X IO5 FFU/ml. The activity of fluids from clone 1, however, fell from 1.4 X lo4 to 1.6 X lo2 FFU/ml between 30 and 60 cell generations. When the typical MSV

1C

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COMMUNICATIONS

morphology was lost focus-forming activity was also lost. A similar ‘Lreversion” has been described by JlacPherson for Rous sarcoma virus transformed hamster BHK21 cells (9). The titration curve for focus-forming activity of virus released by each clone was linear, showing “one-hit” kinetics, over the entire range tested. While Hartley and Rowe (7) have described “two-hit” kinetics for

FIG.

focus formation, other studies using virus obtained from transformed mouse embryo (10) and bovine embryo cells (11) have shown “one-hit” kinetics. This may be due either to focus formation by a “competent” MSV (12,13) that does not require helper activity, to the presence of a large excess of helper, or to aggregates. The present report shows that it is possible

11)

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COMMUNICATIONS

The virus recovered from SV40-transformed 3T3 cells differs from the parent virus in that it has an increased transforming efficiency (14). Whether the properties of the MSV released from Balb/3T3 transformed cells will be similarly altered is currently under study. ACKNOWLEDGMENTS We thank Dr. Janet Hartley for many helpful discussions and Elaine Rands and Lillian Killos for excellent technical assistance. This work was supported by Contract No. PH 43-65-641 from the National Cancer Institute. REFERENCES

DAYS

AFTER

INFECTION

FIG. 2. Rate of cellular DNA synthesis determined by 3H-thymidine incorporation (0.25 &i/ ml, l-hour pulse). A. Uninfected culture; B. infected with 50 FFU/plate; C. infected with 400 FFU/plate. Each culture tested 3 days after last medium change. TABLE FOCUS-FORMING CLONAL

1

ACTIVITY OF FLUIDS FROM LINES OF MS\~-TR~NSFORZVIED BAL~/~T~

Two

Cell generations

% Cells with _ “transformed” morphology

MST’-1

30 60 100

100 510
1.4 1.6

x 10’ x 102 0

nm--3

30 60 100

100 100 100

4.3 2.4 3.2

x 104 X loj X lo4

Clone

FFU/ml

to obtain pure clones of MSV-transformed mouse cells that remain morphologically transformed in culture and continue to release virus even after more than 100 cell generations. The use of a continuous line of non-tumorigenic cells with high cloning efficiency has made it possibleto isolate lines starting from single transformed cells, each of which is the product of an independent virus-cell interaction.

1. AARONSON, S. A. and TODARO, G. J., J. Cellular Physiology 74, 14-148 (1968). 2. AARONSON, S. A. and TODARO, G. J., Science, 162, 1024-1026 (1968). 3. HARTLEY, J. W., personal communication. 4. NAKATA, Y. and BADER, J. P., J. Virology 2, 1255-1261 (1968). 5. HUEBNER, R. J., HARTLEY, J. W., ROWE, W. P., LANE, W. T., and CAPPS, W. I., Proc. N&Z. Acad. Sci. U.S. 56, 116441169 (1966). 6. HARTLEY, J. W., ROWE, W. P., CAPPS, W. I. and HUEHNER, It. J., Proc. Natl. Acad. Sci. U.S. 53:, 931-938 (1965). 7. HARTLEY, J. W. and ROWE, W. P., Proc. Natl. Acad. Sci. U.S. 53, 780-786 (1966). 8. BATHER, Ii., LEONARD, A. and YANG, J., J. Xat!. Cancer Inst. 40, 551-500 (1968). 9. MACPH‘E$~ON, I., Science 148, 1731-1732 (1965) .I ; 10. SIMONS, PI J., DOURMASHKIN, R. R., TURANO, A., PHILLIPS, 1~. E. H., and CHESTERMAN, F. C., Nature 214, 897-898 (1967). 11. THOMAS, XI., BOIRON, M., STOYTCHKOV, Y., and LASNERET, J., Virology 36, 514-518 (1968). lb. O’CONNOR, T. E., and FISCHINGER, P. J., Science 159, 325-329 (1968). 13. BOIRON, M., GUILLEMAIN, B., BERNARD, C., PERIES, J., and CHUAT, J. C., Nature 219, 748-749 (1968). 14. TODARO, G. J., a.nd TAKEMOTO, K. K., Proc. Natl. Acad. Sci. 0’. S., in press. GEORGE J. TODARO STU.~RT A. AARONSON Viral Carcinogenesis Branch National Cancer Institute Bethesda, Maryland 2OOlQ Accepted January 23, 1969