Copyright @ 197X by Academx I’ter\. Inc. All rights of reproduction in any form rese~~cd 0014-4827/79/O Ioo47-llx%o? no/o

Experimental

EFFECTS

Cell Research 118 (1979) 47-54

OF INHIBITORS

TRANSFORMATION INFECTED

IN CHICKEN WITH

JOHN B. HARLEY’ Department

OF LIPID

SYNTHESIS

EMBRYO

ROUS SARCOMA

ON

FIBROBLASTS VIRUS

and HOWARD GOLDFINE

of Tumour Virology, Imperial Cancer Research Fund Laboratories, Lincoln’s Inn Fields, London WCZA 3PX, UK

SUMMARY Cultures of chicken embryo tibroblasts infected with the temperature-sensitive transformation mutant of Rous sarcoma virus (RSV), tsLA24PR-A, were arrested in the cell cycle by incubation at 41°C in a defined medium without serum. After shifting to the permissive temperature, 35”C, these cells assumed the characteristics of transformed cells: They increased their uptake of [3H]2-deoxyglucose, assumed the transformed morphology, and increased the percentage of nuclei labeled with [3H]thymidine. The effects on transformation of 25-hydroxycholesterol, an inhibitor of sterol synthesis, and of cerulenin, an inhibitor of fatty acid synthesis and a partial inhibitor of sterol synthesis, were studied in this system. Cells were able to adopt these characteristics of transformed cells in the presence of 2%hydroxycholesterol. When given 2%hydroxycholesterol 3 h before the temperature shift they sustained the increase in 2-deoxyglucose uptake, but when 25 hydroxycholesterol was given 12 h before the shiftdown from 41°C these cells were subsequently unable to maintain the increased rates of 2-deoxyglucose uptake at 35°C. Cells treated with cerulenin 2 h before the shift also demonstrated only a transient increase in 2-deoxyglucose uptake. We conclude that in this system the early events of transformation can occur in a completely defined medium and that these early events do not require normal rates of sterol synthesis.

Several manifestations of the transformation of cells by the avian tumor viruses are related to the structure and function of the cell surface and cytoplasmic membrane. These include increased transport and altered permeability of a variety of solutes, alterations of glycolipids and glycoproteins in the cytoplasmic membrane, increased agglutinability by lectins, changes in the densities of lectin-binding sites, changes in external proteins, and decreased adhesiveness (reviewed by Robbins & Nicolson [l]). In avian tumor virus-transformed chicken embryo cells [2, 31 and in cells transformed by SV40 [4, 51, changes in the ratios of oleic acid to arachidonic acid in cellular lipids have been observed. Until recently it has 4-781802

not been possible to study the effects of prior modification of membrane lipid composition on the process of cell transformation induced by tumor viruses. Recently, several laboratories have investigated methods for controlling the access of cells in culture to precursors of lipid synthesis. The basis for these methods is the growth of cells in media containing lipid-free or lipid-poor sera, which compels the cells to synthesize fatty acids and sterols de novo or to incorporate specific fatty acids or ’ Present address: Department of Internal Medicine, Yale University, New-Haven, CT 06.510,USA. 2 Present address: Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Et-p Cell Rr, 118 (1979)

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Harley and Goldjbe

sterols added to the media. The cells also become dependent upon the presence of choline in the medium and several laboratories have taken advantage of this requirement to investigate the consequences of replacing choline with its analogues (reviewed by Horwitz [6]). The discovery of two inhibitors of membrane lipid synthesis has provided an additional tool for studying effects of lipid deprivation. Several oxygenated sterol derivatives have been shown to inhibit cholesterol synthesis in animal cells [7,8] and cerulenin has been shown to inhibit fatty acid synthesis in bacteria, fungi [9], the slime mold Dictyostelium discoideum [lo] and in chicken embryo cells [ 111.Cerulenin also partially inhibits sterol synthesis in eukaryotic cells [9-111. We have investigated the effects of these inhibitors of lipid synthesis on early events in the transformation of chicken embryo cells by a temperature-sensitive mutant of Rous sarcoma virus (RSV). MATERIALS

AND METHODS

Chicken embrvo fibroblast cultures of C/E ohenotvne were prepared from Brown Leghorn embryos (Wickham Laboratories, Hants) by standard techniques f121, and grown in Dulbecco’s modified Eagle’s~medium (DMEM) supplemented with 10% tryptose phosphate broth, 5% calf serum and 1% chick serum (complete medium). Secondary cultures were infected with the RSV mutant rsLA24PR-A at a multiplicity of 0.5-I .O focus-forming unit/cell and incubated for 4 days. This virus is temperature-sensitive in a function continuously required for the maintenance of cell transformation: the uermissive temuerature is 35°C and the restrictive is 41°C [ 131.Cultures were then incubated and subcultured as described bv Bell et al. fl41, exceot that 3rd passage cells were kept at 35°C. For later measurement of [3H]2-deoxyglucose uptake, the fourth passage was made into 3.5 cm diameter dishes with 5X IO5 cells/dish in DMEM plus biotin, 1.5 PgJml and incubated at 41°C. After additions of inhibitors cultures were shifted to 35°C except for controls, at the times indicated below. For autoradiography of [3H]thymidine-labeled cells, the fourth passage was made with complete medium into the 1 cm diameter wells of Linbro travs. at 3~ 104cells or 5x IO4 cells/well. After 1 day these cells were washed twice with approx. 0.5 ml DMEM and maintained in this medium for 2 days at 41°C. Exp Cell Re.\ I18 f 1979)

600

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Fig. I.

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Abscissa:

cr,.,~Xtam~.

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time (hours);

ordinarr:

no. of cells/

Effect of 25-hydroxycholesterol on the growth of chick embryo fibroblasts. Cultures were plated in 3.5 cm dishes in DMEM supplemented with 20% delipidated FCS plus 1.5 &ml biotin. At the time of plating, the following concentrations of 25-hydroxycholesterol were added (in pg/ml): A, none; 0, 0.05: 0, 0.10; Cl, 0.25; W, 0.5; V, 1.0; v, 2.5. Cells were counted in a hemocytometer after removal with 0.05 % trypsin (Difco, Bacto). Data represent the means of duplicate cell counts of a representative experiment.

Uptake of 2deoxyghrcose was measured according to Weber [15]. Cultures were washed once with room temperature phosphate-buffered saline (PBS) and incubated with 0.5 &i of [l-3H]2deoxy-o-glucase. 250 uM. for 9 min at room temperature (21°C). After four washings with 1 ml ice-coid PBS, 1’ ml of 5 % trichloroacetic acid (TCA) was added, the dishes were shaken in a tray at least.50 times, and 0.5 ml of the extract was counted in 4.5 ml Aquasol II (New England Nuclear). Protein content was determined by the method of Lowry et al. [16] after washing the plates three times with methanol and solubilizing the cells in 0.2 ml 1 N NaOH. For determination of nuclear labeling by [3H]thymidine, cultures were incubated with 0.8 &i of [Me3H]thymidine, 1.6 PM, for varying periods of time at 35 or 41°C. They were then washed twice with apurox. 1 ml ice-cold PBS. twice with I ml ice-cold 5% TCA and twice with methanol. The first TCA wash was left on the cells for at least 2 min. The cells were then covered with Ilford Nuclear Research Emulsion KS for 4 days at room temperature. The trays were developed in Kodak D-19 developer, fixed, washed with methanol, stained lightly in Giemsa, and examined for labeled nuclei. flJ4ClAcetate incoruoration into sterols and fattv acids was measured as described by Kandutsch & Saucier [ 181with slight modification [ 111.The ratio of counts in the sterol fraction relative to counts in fatty

Lipid synthesis and transformation 2.0

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Fig. 2. Abscissa: time after temperature shift (hours); &inure: 2-deoxyglucose uptake in nmoles/min/mg protein. Effect of 25hydroxycholesterol added 3 h before the temperature shift on uptake of [l-3H]2-deoxyglucase by cells made stationary in serum-free medium at 41°C. -, Cultures shifted to 35°C; ---, cultures kept at 41°C. 0, 25Hydroxycholestero1, 2.5 pg/ml in a BSA and ethanol-containing solution added 3 h before temperature shift. 0, BSA, 1.13 mgfml, and ethanol, 2.5 PI/ml, added 3 h before temperature shift; 0, no additions. Points represent the mean? S.E. from 2 expts in which each measurement was done in duplicate.

49

RESULTS Effect of 25hydroxycholesterof on the growth of chick embryofibroblasts Cells grown in DMEM supplemented with 5-20% delipidated FCS plus biotin, 1.5 pg/ml, grew as well as cells grown with 20% FCS or standard complete medium containing 5 % calf serum, 1% chick serum, and 10% tryptose phosphate broth (data not shown). Similar results have recently been reported by Hale et al. [20]. The addition of increasing amounts of 25hydroxycholesterol to uninfected cells at the time of plating resulted in complete inhibition of growth after 24 h with 1.0 pg/ml of inhibitot-, as shown in fig. 1. During the first 24 h the cell number had increased approx. 2fold in the inhibited cultures and 3-fold in uninhibited cultures. Similar effects of 25hydroxycholesterol on the growth of mouse L cells have been reported [ 171.

Effect of 25hydroxycholesterol on sterol synthesis [ 1-‘*C]Acetate incorporation into sterols acids was used to measure the specific effect of 25- and fatty acids was measured during three hydroxycholesterol on sterol synthesis [7]. Similar retime periods after addition of 2.5 pglml insubs were obtained by directly comparing incorporation of acetate into sterols alone in inhibited and con- hibitor to RSV tsLA24-infected chick emtrol cultures, since incorporation into fatty acids was bryo fibroblasts and done in parallel with not greatly affected over the time-interval studied. 25-Hydroxycholesterol was obtained from Steralthe transport assays described below. Duroids Inc. (Wilton, N.H., USA) and recrystallized once from ethanol. Sterols were dissolved in ethanol and ing the period 3-9 h after addition, acetate diluted with 9 vol of 5 % bovine serum albumin (BSA) incorporation into sterols was inhibited an in 0.14 M NaCl prior to addition to cultures [7]. Conaverage of 85 %; during the period from 12 trols contained the same amounts of ethanol and BSA as the experimental cultures. Cerulenin was the gener- to 18 h after 25-hydroxycholesterol addious gift of Dr S. Omura. It was dissolved in absolute alcohol, stored at -7o”C, and diluted with 4 vol of tion, acetate incorporation into sterols was double-distilled water prior to addition to cultures. inhibited an average of 93 %; and during Controls had the same volume of absolute ethanol as the period from 9 to 27 h after addition of the experimental cultures. Labeled compounds were purchased from the Ra- the inhibitor, acetate incorporation was indiochemical Centre (Amersham, Bucks., UK). Dehibited by an average of 88%. lipidated fetal calf serum (FCS) was prepared by

ethanolether extraction as described by Horwitz et al. [5] except that the same volumes of solvents were used on 100 ml of serum [19]. The dried residue was reconstituted in saline to 80 mg/ml, which is about twice the concentration of protein in FCS. Thus 5% v/v of the reconstituted delipidated serum is the equivalent of 10% whole serum in protein content and is referred to in this paper as 10% delipidated serum.

Effects of 25hydroxycholesterol on the enhancement of deoxyglucose uptake by transformed cells As shown by Bell et al. [14], chick embryo fibroblasts infected with RSV tsLA24 and E.t-p Cd/ Res ii8 (19791

50 4.0

Harley and Goldfine

r

I

1

I

I

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2 4 6 8 IO 12

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4. Abscissa: time after the temperature shift (hours); ordinate: 2-deoxyglucose uptake in nmolesl min/mg protein. Effect of cerulenin added 2 h before temperature shift on uotake of 2deoxvelucose bv cells made stationary in’serum-free me&m at 4i”C. -, Cultures shifted to 35°C; ---, cultures kept at 41°C. 0, Cerulenin, 12.5 @g/ml; Cl, control, 25 ~1 of absolute ethanol-water ((1 : 4) v/v). All additions were made 2 h before the temperature shift. Data presented as in fig. 2.

Fig. 0 2 4 6 810

12

24

Fig. 3. Abscissa: time after temperature shift (hours); ordinate: 2deoxyglucose uptake in nmoles/min/mg

protein. Effect of 25hydroxycholesterol added 12 h before the temperature shift on uptake of 2-deoxyglucose by cells made stationary in serum-free medium at 41°C. -, Cultures shifted to 35°C; - --, cultures kept at 41°C. 0, 25Hydroxycholestero1, 2.5 pg/ml, in a BSA plus ethanol-containing solution; 0, 25hydroxycholesterol, 2.5 pg/ml and cholesterol, 25 pg/ml, both in BSA plus ethanol-containing solutions; A, control for 25-hydroxycholesterol addition containing BSA, 1.13 mg/ml and ethanol, 2.5 pi/ml; 0, control for 25-hydroxycholesterol plus cholesterol additions containing BSA, 3.4 mg/ml and ethanol, 7.5 pi/ml. All additions were made 12 h before the temperature shift. Points represent the mean + S.E. from 2 expts in which each measurement was done in duplicate.

maintained in DMEM supplemented with 10% tryptose phosphate broth for 2 days, assume a transformed morphology and increase their uptake of 2-deoxyglucose approx. 5-fold by 24 h after a shift from 41 to 35°C. The increase in deoxyglucose uptake took place after a lag of 4 h. In the present experiments, cells were maintained for 1 day in DMEM without tryptose phosphate broth. Control tsLA24-infected cells with BSA plus ethanol added, increased deoxyglucose uptake rates approx. 3.5-fold within 24 h after the temperature shift and Exp Cell Rrs 118 (1979)

assumed a transformed morphology. Control cells with no BSA and ethanol increased the deoxyglucose uptake rate approx. 2-fold and also changed morphology. In both cases the increase in uptake occurred without a lag as seen in fig. 2. During the same period, cells maintained at 41°C showed essentially no change in deoxyglucose uptake, as expected. In cultures to which 25-hydroxycholesterol in a BSA plus ethanol solution had been added 3 h prior to the temperature shift, the increase in deoxyglucose uptake also occurred without lag, and for the first 6 h was as great as in the control cultures. At 24 h deoxyglucose uptake was essentially the same in 25-hydroxycholesterol-treated cultures as in the controls without BSA and ethanol, but less than in the controls containing BSA and ethanol. Some inhibition of deoxyglucose uptake was seen in 25-hydroxycholesteroltreated cells maintained at 41°C.

Lipid synthesis and transformation

51

crease in uptake of the cultures exposed to 25hydroxycholesterol plus cholesterol was slightly delayed. Effect of cerulenin on deoxyglucose uptake Cerulenin, 12.5 pg/ml, inhibits [lJ4C]acetate incorporation into fatty acids by 90% and into sterols by 47 % 2. h after addition to uninfected chickembryo cells [ 111.Simi0 2 4 6 8 IO 22 lar results were obtained with RSV tsLA24infected cells. The effect of cerulenin added time after the temperature shift Fig. 5. Abscissa: (hours); ordinate: % of labeled nuclei. 2 h before the temperature ‘shift on the inEffect of 2ShydroxycholesteroI added 3 h before a temperature shift on accumulated nuclear incorporacrease in deoxyglucose uptake was extion of r3H]thymidine by cells made stationary in se- amined. As shown in fig. 4, an enhanced rum-free medium at 41°C. -, Cultures shifted to 35°C; rate of uptake of deoxyglucose uptake was ---1 cultures kept at 41°C. n , 0, 25Hydroxycholesterol, 2.5 &ml; 0, 0, control for 25hydroxychoseen in cells shifted to 35°C in the presence lesterol addition containine BSA. 1.13 me/ml and ethanol, 2.5 &ml. Addition o? inhibitor pluscontrol solu- of 12.5 pg/ml cerulenin for 4 h after the tions was 3 h before the temnerature shift. PHlThvmitemperature shift. After that the rate of dine was added at the time of the shift. Data presented deoxyglucose transport in the cerulenin as in fig. 2. treated cultures declined to the level of cells kept at 41°C. Further enhancement of deThe capacity of these cells to recover oxyglucose uptake was seen in control culfrom exposure to 2.5 pg/ml 25hydroxytures for up to 24 h after the temperature cholesterol was assessed as an extension of shift. the above experimental protocol. After growth arrest in DMEM, and a 27 h ex- Effect of 25-hydroxycholesterol and posure to the inhibitor, the cells were cerulenin on uptake of [3H]thymidine washed and the medium was replaced with The majority of chick embryo fibroblasts complete medium. Cells which had been infected with RSV tsLA24 are arrested in kept at 41°C recovered and grew, while growth between mitosis and the completion those which had been shifted to 35°C 3 h of S phase when they are placed in medium after addition of 25-hydroxycholesterol did without serum and kept at the restrictive not grow when returned to complete me- temperature, 41°C [14]. On shift to 35°C dium. these cells transform, as shown by the enAs shown in fig. 3, when 25-hydroxyhanced deoxyglucose uptake, and the incholesterol was added 12 h before the tem- creased synthesis of DNA [14]. The cell perature shift from 41 to 35°C increased number increases approx. 60% at 35°C and deoxyglucose uptake was seen for 4 h after only 10% at 41°C in 29 h [14]. When similar the shift, but by 8 h the rate of uptake experiments were performed in the presdeclined to the level of cultures maintained ence of 25-hydroxycholesterol, we obat 41°C. Controls increased their rate of served no inhibition of r3H]thymidine ladeoxyghrcose uptake approx. 3-fold by 24 h belling of nuclei at either 35 or 41°C. At after the temperature shift though the in- both temperatures, 25hydroxycholesterolExp Cell Res I18 f 1979)

52

Harley and Goldfinr

treated cells had equal or higher labeling indices as seen in fig. 5. Similar results were obtained with cells kept in DMEM plus 10% tryptose phosphate broth, but the labeling indices were higher. At 22 h they were 52?3.4% and 47+10% for cells shifted to 35°C with and without 25hydroxycholesterol respectively; and 3 l? 1.1% and 29+3.3% for cells kept at 41°C with and without 25hydroxycholestero1, respectively (averages of two separate experiments). These labeling indices were lower than those found by Bell et al. in DMEM plus 10% tryptose phosphate broth [14], which we believe is partly the result of our using a lower specific activity of [3H]thymidine. Similar experiments were done with cells exposed to cerulenin, 15 pglml. The results were somewhat erratic because cells treated with cerulenin for 26 h became detached from the dish and many were lost before or during the washing procedure. However, relatively few of the cells that remained attached had labeled nuclei 24 h after the temperature shift in the presence of cerulenin compared to control cultures which had from 24 to 62% labeled nuclei at 35°C and from 20 to 32 % labeled nuclei at 41°C. DISCUSSION Simplifying the media in which transformation studies are performed should help elucidate the nutritional and biochemical requirements for transformation. Bell et al. have shown that chicken embryo fibroblasts infected with a transformation mutant of RSV, tsLA24, were arrested between mitosis and S phase by exposure to serum-free medium containing tryptose phosphate broth at 41°C for 2 days. These cells assumed a transformed morphology, increased their rate of 2-deoxyglucose upErp Cdl Res II8 (1979)

take, and entered S phase when they were shifted to the permissive temperature. 35°C [14]. We have shown that these cells in a completely defined medium also exhibit the same manifestations of the transformed state. In this study we have investigated the relationship between lipid synthesis and transformation using inhibitors of sterol and fatty acid synthesis. 25-Hydroxycholesterol at 2.5 pug/ml inhibited sterol synthesis by approx. 85-90%. When the inhibitor was added 3 h before a shift to the permissive temperature, RSV tsLA24-infected chick embryo fibroblasts proceeded to increase their uptake of 2-deoxyglucose (fig. 2), to assume the transformed morphology, and to increase the proportion of nuclei labeled with [3H]thymidine, all in a similar fashion to untreated control cells and in contrast to cells maintained at the non-permissive temperature. We conclude that normal rates of de novo sterol synthesis are not required for the early manifestations of cellular transformation by RSV. When infected cells were treated with either 25hydroxycholesterol starting 12 h before the temperature shift or with cerulenin 2 h before the shift, they were initially able to increase their uptake of 2-deoxyglucase as were the untreated control cells. They were, however, incapable of maintaining this enhanced rate and the rate of 2-deoxyglucose uptake decreased to the level of the controls at the non-permissive temperature (figs 3,4). Beyond the obvious considerations of inhibitor toxicity and population heterogeneity these observations are also consistent with a requirement for continued sterol or fatty acid synthesis to maintain this property of the transformed state. We have shown elsewhere that cerulenin, but not 25-hydroxycholesterol, inhibits the

Lipid synthesis and transformation

replication of RSV and that cleavage of pr76, the precursor of the non-glycosylated viral structural proteins ~27, ~19, ~15, and ~12, is inhibited in cerulenin-treated cells [ 111.In addition, the effects of cerulenin on [3H]thymidine incorporation and on RSV replication are not reversible by either mixtures of fatty acids and cholesterol or by whole serum. Therefore, the effects of cerulenin on non-lipid constituents may prevent continued expression of transformation. Presumably the plasma membrane of 25 hydroxycholesterol-treated cells is progressively depleted of sterols, as has been demonstrated in other experimental systems [17, 191.When the plasma membrane, or other lipid-containing structures, becomes sterol-depleted at 41°C the ability of the cell to sustain the enhanced 2-deoxyglucase uptake at the transformed state is lost (fig. 3). On the other hand, the cells maintained at 41°C were able to survive and recover after 25hydroxycholesterol exposure, while those shifted to 35°C were not. These results imply that there are steroldependent differences between cells at the permissive and non-permissive temperatures as a consequence either of a differential temperature-dependent inhibitor toxicity or of an inherent difference between the transformed and non-transformed state . The ability of RSV tsLA24-infected cells to enter S phase when shifted to the permissive temperature in serum-free medium in the presence of 25-hydroxycholesterol is unexpected. Mouse lymphocytes stimulated with PHA undergo a cycle of sterol synthesis followed by DNA synthesis. Both are blocked by the addition of 25-hydroxycholesterol prior to the peak of the cycle of sterol synthesis. These findings prompted Chen et al. [21] to postulate that sterol syn-

53

thesis was needed in this system for the cells to enter S phase. Cornell et al. [19] have shown that both human fibroblasts (WI38) and rat myogenic cell lines arrest reversibly in Gl when sterol synthesis is inhibited by 25-hydroxycholesterol, when fatty acid synthesis is blocked by removal of biotin, or when cells are deprived of choline. Specific reconstitution allows these cells to pass through the cell cycle. Hatten et al. found that transformed SV lOl-3T3 cells, unlike 3T3 cells, did not arrest in any particular stage of the cell cycle, even though the increase in cell numbers came to a halt, when cells were transferred to a medium containing lipid-depleted serum without fatty acid or biotin [22]. The inability of 25-hydroxycholesterol to prevent RSV tsLA24-infected chick embryo cells from entering S phase raises additional questions that suggest avenues of future investigation. We wish to thank Jane Sandal1for technical assistance and John Wyke, Jennifer Beamand and Celia Norris for helpful advice. J. B. H. was a recipient of a National Institute of Allergv and Infectious Diseases Postdoctoral Fellowship cl-F32-AI-05186). H. G. was a recipient of a Josiah Macv Foundation Scholar Award, 197677.

REFERENCES 1. Robbins, J C & Nicolson, G L, Cancer a comprehensive treatise (ed F F Becker) vol. 3. chaot. 21. Plenum Press, New York (1975). 2. Yau, T M, Buckman, T, Hale, A H & Weber, M J, Biochemistry 15 (1976) 3212. 3. Hale, A H, Yau, T M & Weber, M J, Biochim biophys acta 443 (1976) 618. 4. Howard, B V & Kritchevsky, D, Int j cancer 4 (1%9) 393. 5. Horwitz, A F, Hatten, M E & Burger, M M, Proc natl acad sci US 71 (1974) 3115. 6. Horwitz, A F, Growth, nutrition, and metabolism of cells in culture (ed G H Rothblat & V J Cristofalo) vol. 3. Academic Press, New York (1977). 7. Kandutsch, A A & Chen, H W, J biol them 248 (1973) 8408. 8. y Ibid 249 (1974) 6057. 9. Omura, S, Bact rev 40 (1976) 681. 10. Chance, K, Hemmingsen, S &Weeks, G, J bacteriol 128 (1976) 21. 11. Goldfine, H, Harley, J B & Wyke, J A, Biochim biophys acta 512 (1978) 229. E.rp Cdl Rrs 118 (I 979)

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12. Vogt, P K, Fundamental techniques in virology (ed K Habel & N P Salzman). Academic Press, New York (1969). 13. Wyke, J A & Linial, M, Virology 53 (1973) 152. 14. Bell, J G, Wyke, J A & Macpherson, I A, J gen virol 27 (1975) 127. 1.5. Weber, M J, J biol them 248 (1973) 2978. 16. Lowry, 0 H, Rosebrough, N J, Fan-. A L & Randall, R J, J biol them 193 (1951) 265. 17. Kandutsch, A A & Chen. H W, J biol them 252 (1977) 409. 18. Kandutsch. A A & Saucier. S E. J biol them 244 (1969) 2299.

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19. Cornell, R. Grove, G L, Rothblat, G H & Horwitz. A F. Exp cell res 109 (1977) 299. 20. Hale, A H, Pessin, J E. Palmer, F, Weber. M J & Glaser, M, J biol them 252 (1977) 6190. 21. Chen, H W, Heiniger, H-J & Kandutsch. A A. Proc natl acad sci US 72 (1975) 1950. 22. Hatten. M E, Horwitz, A F & Burger, M M, Exp cell res 107 (1977) 31. Received May I I, 1978 Revised version received August 25, 1978 Accepted August 31. 1978

Effects of inhibitors of lipid synthesis on transformation in chicken embryo fibroblasts infected with Rous sarcoma virus.

Copyright @ 197X by Academx I’ter\. Inc. All rights of reproduction in any form rese~~cd 0014-4827/79/O Ioo47-llx%o? no/o Experimental EFFECTS Cell...
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