Vol. 45, No. 2 JOURNAL OF VIROLOGY, Feb. 1983, p. 895-897 0022-538X/83/020895-03$02.00/0 Copyright C 1983, American Society for Microbiology Persistence of Interferon Action in Mouse Cells Permeabilized with Lysolecithin ELSBETH J. LEEt AND THAZEPADATH SREEVALSAN* Department of Microbiology, Georgetown University, Schools of Medicine and Dentistry, Washington, D.C. 20007 Received 19 August 1982/Accepted 16 September 1982 DNA synthesis, as measured by incorporation of [3H]TTP, was inhibited in Swiss mouse 3T3 cells treated with interferon and subsequently permeabilized with lysolecithin. The degree of inhibition observed was similar in intact or permeabilized cells. The interferon-induced antiviral state was retained in per- meabilized cells. Interferon (IFN) induces pleiotropic effects on mammalian cells, including the inhibition of DNA synthesis and cell growth (7). The mecha- nisms mediating these effects, however, remain poorly defined at present. Incorporation of exo- geneously added [3H]thymidine into acid-insolu- ble material is the usual technique employed for the determination of DNA synthesis in mamma- lian cells. Using this technique, several authors have shown that there is significant inhibition of the incorporation of labeled thymidine into cel- lular DNA in IFN-treated cells (1, 5, 6, 8). In experiments with L1210 cells, however, Brouty- Boye and Tovey (2) have reported that IFN inhibits the uptake of thymidine into cells, a finding which has been confirmed with Swiss mouse 3T3 cells (T. Sreevalsan, unpublished data). Therefore, a reduction in uptake could account for the observed inhibition in the incor- poration of [3H]thymidine into acid-insoluble material in Swiss 3T3 cells treated with IFN (1, 6). In this report, we examined the effect of IFN on DNA replication in permeabilized cells, into which [3H]TTP can be introduced directly. If the mechanism of inhibition of DNA synthesis is dependent on the effect of IFN on the ability of cells to take up thymidine, one would expect the incorporation of [3H]TTP into cellular DNA in permeabilized cells to be unaffected by IFN. Our results indicated that the inhibitory effects of IFN on DNA synthesis persisted even after the cells were permeabilized. The procedure used to permeabilize Swiss 3T3 cells was a modification of a method de- scribed recently (3, 4). Cells to be permeabilized were seeded in 33:mm dishes at 105 cells per dish. When cells were confluent and quiescent, t Present address: Laboratory of Tumor Virus Genetics, National Cancer Institute, National Institutes of Health, Be- thesda, MD 20205 cultures were stimulated for 18 h with Dulbecco modified Eagle medium containing 10% calf serum as described previously (6). Unstimulated cultures received medium containing no serum. The cells were permeabilized by using lysoleci- thin, and DNA synthesis was measured by the incorporation of [3H]TTP into the acid-insoluble fraction as described by Castellot et al. (3). Preliminary experiments indicated that, com- pared with unstimulated cells, there is an in- crease (50- to 100-fold) in the ability of stimulat- ed cells to synthesize DNA. Additionally, [3H]TTP incorporation was linear up to 20 min after permeabilization of the cells. Thereafter, the ability of permeabilized cells to synthesize DNA was gradually lost. These findings were similar to those reported previously (3, 4). The results in Table 1 show that incorporation of [3H]thymidine by intact cells or [3H]TTP by permeabilized cells was impaired in mouse IFN- treated cultures. The degree of inhibition of DNA synthesis was similar in both intact and permeabilized cells. There was some (ca. 20%) reduction of DNA synthesis when cells were treated with human lymphoblastoid IFN rather than mouse IFN (results not shown). This was not surprising since it is now known that human IFN can act in cells of heterologous species, including mice (9). The results presented in Fig. 1 show that the observed inhibitory effect of mouse IFN on DNA synthesis was dose depen- dent. Again, comparable levels on inhibition were observed in intact or permeabilized cells at a given dose of IFN. Experiments were performed to determine whether the antiviral state induced by IFN in cells could persist after permeabilization. The results (Table 2) showed that the IFN-induced antiviral state, as judged by resistance to en- cephalomyocarditis virus replication, was re- 895 896 NOTES TABLE 1. Inhibitory effect of IFN on DNA synthesis in intact or permeabilized 3T3 cellsa Radioactivity in: Treatment ~Intact cells Permeabilized Treatment (CpM Xcells cells (cpm x 106 cells) 104/Mg Of protein) Unstimulated 0.04 ± 0.01 0.05 ± 0.007 Serum 1.9 ± 0.09 3.84 ± 0.006 Serum + mouse 0.35 ± 0.02 0.58 ± 0.16 IFN a Quiescent Swiss mouse 3T3 cells in 33-mm Nunc dishes were prepared and then stimulated with fresh medium containing serum as described previously (6). Mouse IFN (specific activity, 2 x 105 U/mg of pro- tein), prepared by described methods (6), was used at 1,000 U/ml. The cultures were incubated for 18 h, at which time DNA synthesis was ascertained. Cultures to be permeabilized were chilled by placing the dishes on a metal tray in ice and then washed two times with cold phosphate-buffered saline (pH 7.4) containing 2 mM CaC12. Dishes were then washed with cold buffer containing 35 mM MgCI2 and 1 mM CaC12. Monolay- ers were permeabilized in the same buffer (1 ml) with 60 to 80 ,ug of lysolecithin (Sigma Chemical Co., St. Louis, Mo.) per ml for 2 min at 0°C. (The concentra- tion of lysolecithin required for optimal permeabiliza- tion [>95%] varied depending on the batch of cells used. Therefore, for each set of experiments the optimum concentration of lysolecithin required was determined.) The degree of permeabilization was as- sessed by determining the uptake of trypan blue by the cells. After removal of the overlying solution, permea- bilized monolayers were incubated with the buffer described above (1 ml), without CaC12, containing 0.25 mM dCTP, 0.25 mM dGTP, 0.25 mM dATP, 0.12 mM GTP, 0.12 mM UTP, 0.12 mM CTP, 1.25 mM ATP, 10 mM phosphoenolpyruvate, and 5 ,uCi/ml of 60 ,uM [methyl-3H]TTP tetrasodium salt (specific activity, 77.4 Ci/mmol; New England Nuclear Corp., Boston, Mass.). Intact cultures were incubated for 30 min with 0.25 ,uCi of 1 t±M [3H]thymidine (specific activity, 52 Ci/mmol, New England Nuclear Corp.). Permeabi- lized cells were incubated for 20 min at 37°C in humidified 10% CO2. Monolayers were washed with cold phosphate-buffered saline containing 2 mM CaC12, followed by a 15-min incubation with cold 5% trichloroacetic acid containing 0.2 M sodium PPi. Cultures were washed five times with cold 5% tri- chloroacetic acid and then washed three times with absolute ethanol. The air-dried monolayers were dis- solved in 0.1 N NaOH at 37°C for 30 min, and the amount of radioactivity was determined by liquid scintillation counting in acidified Ready Solv (Beck- man Instruments, Inc., Palo Alto, Calif.). Values represent the mean ± the standard error of the mean of three experiments. tained even after the cells were permeabilized with lysolecithin. Although the synthesis of en- cephalomyocarditis virus-specific RNA in con- trol permeabilized cells was low compared with that in intact cells, there was a significant reduc- 100r- -J 0 c- z 0 LL 0 0 -J w 60k 401- 20k 10 F~~~~~~~~V 100 1000 UNITS OF IFN FIG. 1. Inhibitory effect of IFN on DNA synthesis in permeabilized cells. Quiescent cultures (33 mm) were prepared and stimulated as described in Table 1. Some of the cultures received mouse IFN (specific activity, 2 x 105 U/mg of protein) at the indicated concentration. After 18 h of incubation, sets of cul- tures were permeabilized, and DNA synthesis in cells was measured by methods identical to those described in Table 1. Values are expressed as a percentage of the yield of the control stimulated cells and represent the mean the standard error of the mean of three experiments. Symbols: O, intact cells; 0, permeabi- lized cells. TABLE 2. Persistence of IFN-induced antiviral activity in permeabilized 3T3 cells' Radioactivity (cpm x 103 of [3H]uridine per culture Treatment per 45 min) in: Intact Permeabilized cells cells Unstimulated 5.10 2.30 Serum 43.30 17.70 Serum + actinomycin D 0.56 0.12 Serum + actinomycin D 10.70 1.90 + virus Serum + actinomycin D 0.24 0.07 + virus + IFN a Quiescent cultures (33 mm) were prepared and stimulated as described in Table 1. Wherever indicat- ed, cultures received mouse IFN (1,000 U/ml) at the time of stimulation. After approximately 15 h of incu- bation, cultures were challenged with encephalomyo- carditis virus and subsequently incubated for 6 h in media containing 2% dialyzed calf serum. Cultures receiving 1 p.g of actinomycin D per ml were incubated with the drug at this time. After incubation, cultures were permeabilized as described in Table 1. This was followed by a 45-min pulse with [3H]uridine (1 ,uCi; specific activity, 77 Ci/mmol). Cell-specific (no actino- mycin D) or virus-specific (plus actinomycin D) acid- insoluble counts were expressed as counts per minute incorporated per culture. J. VIROL. NOTES 897 tion in the amount of viral RNA synthesis in IFN-treated cultures. These results suggest that the antiviral activity induced by IFN in cells can persist even after the cells are permeabilized. The major metabolic pathway for the de novo synthesis of thymidylic acid in cells involves the transfer of the methylene group from N5,N10 methylene-tetrahydrofolic acid to position 5 of deoxyuridylic acid and is catalyzed by the en- zyme thymidylate synthetase. The salvage path- way involves the transfer of the -y-phosphate of ATP to the 5' position of thymidylic acid and is catalyzed by the enzyme thymidine kinase. The present results clearly show that introduction of TTP directly into cells did not abrogate the inhibitory effects of IFN on DNA synthesis. This suggests that the anti-mitogenic effect of IFN should reside, at least partly, on mecha- nisms other than the reduced uptake of thymi- dine. Our results also show that the antiviral activity induced by IFN in cells persisted even after the cells were permeabilized. This suggests that the factors mediating the inhibitory effects of IFN are molecules which may be tightly bound intracellularly. This work was supported by grant CD#56 from the Ameri- can Cancer Society. LITERATURE CITED 1. Balkwill, F. R., and J. Taylor-Papadimitriou. 1978. Inter- feron affects both G0 and S+G2 in cells stimulated from quiescence to growth. Nature (London) 274:798-800. 2. Brouty-Boye, D., and M. G. Tovey. 1978. Inhibition by interferon of thymidine uptake in chemostat cultures of L1210 cells. Intervirology 9:243-252. 3. Castellot, J. J., Jr., M. R. Miller, D. M. Lehtomaki, and A. B. Pardee. 1979. Comparison of DNA replication and repair enzymology using permeabilized baby hamster kid- ney cells. J. Biol. Chem. 234:6904-6908. 4. Miller, M. R., J. J. Castellot, Jr., and A. B. Pardee. 1978. A permeable animal cell preparation for studying macromo- lecular synthesis. DNA synthesis and the role of deoxyri- bonucleotides in S phase initiation. Biochemistry 17:1073- 1080. 5. Sokawa, Y., Y. Watanabe, and Y. Kawada. 1977. Suppres- sive effects of interferon on the transition from a quiescent to a growing state in 3T3 cells. Nature (London) 268:236- 238. 6. Sreevalsan, T., E. Rozengurt, J. Taylor-Papadimitriou, and J. Burchell. 1980. Differential effect of interferon on DNA synthesis, 2-deoxyglucose uptake and ornithine decarbox- ylase activity in 3T3 cells stimulated by polypeptide growth factors and tumor promoters. J. Cell. Physiol. 104:1-9. 7. Taylor-Papadimitriou, J. 1980. Effect of interferon on cell growth and function, p. 13-46. In I. Gresser (ed.), Interfer- on 1980. Academic Press, Inc., New York. 8. Tovey, M. G., D. Brouty-Boye, and I. Gresser. 1975. Early effect of interferon on mouse leukemia cells cultivated in a chemostat. Proc. Natl. Acad. Sci. U.S.A. 72:2265-2269. 9. Weck, P. K., E. Rinderknecht, D. A. Estell, and N. Steb- bing. 1982. Antiviral activity of bacteria-derived human alpha interferon against encephalomyocarditis virus infec- tion in mice. Infect. Immun. 35:660-665. VOL. 45, 1983