Effect of Rous Sarcoma Virus Infection on Recovery of Nucleolar RNA, Ribonucleoprotein, and DNA Synthesis from Inhibition by Actinomycin D'

[CANCER RESEARCH 29, 1598-1605, August 1969]

R. Gerald Suskind, Thomas W. Pry, and Giancarlo F. Rabotti: Laboratory of Experimental Pathology, National Institute of Arthritis and Metabolic Disease, and Viral Biology Branch, National Cancer Institute, NIH, Bethesda, Maryland 20014

SUMMARY Rous sarcoma virus (RSV) remains poorly understood, and relatively little is known about the site and manner of viral The effect of Rous sarcoma virus (RSV) infection on the RNA synthesis (15, 16, 23-26, 32, 33, 38-40; S. W. C. Fiske recovery of nucleolar RNA and protein synthesis from selec- and F. Gaguenau, unpublished data). Studies with metabolic tive inhibition by actinomycin D was investigated using radio- inhibitors of RNA synthesis, such as actinomycin, and inhibi- autographic technics. A transient increase in the grain count tors of DNA and protein synthesis have shown a complex over nucleoli of actinomycin-treated cells, pulse-labeled with interdependence of infectivity and transformation with cellu- tritiated uridine, was observed in cells 10 to 18 hours after lar DNA and protein synthesis (2-4, 14, 20, 38, 39, 42, 43); infection with Bryan strain RSV. The proportion of the total however, the mechanism of inhibition is still incompletely RNA synthesized in the nucleolus at that time is greater than understood. Early administration of actinomycin results in in- in infected or uninfected cells not treated with actinomycin. hibition of cellular transformation (4, 38, 42) and will prevent In mock-infected cells, or in cells exposed to inactivated virus, RNA labeling of the purified virus particle (23). It is unclear, inhibition of nucleolar RNA synthesis persists for 24 hours however, whether this results from inhibition of intracellular after removal of the inhibitor. During this period, actinomycin viral RNA synthesis (39) or from host-dependent functions inhibits both the accumulation of protein label in the nucleo- necessary in the assembly of the viral RNA (23). Resolution of lus as well as cellular DNA synthesis, and this inhibition is this question by the use of physical separation technics is com- identical in infected and in uninfected cells. However, 48 to 72 plicated by the fact that the amount of intracellular, viral hours after infection, a period coincident with the appearance RNA is minuscule in comparison with that of host RNA and of some transformed cells, DNA synthesis is significantly great- that viral RNA appears to be rapidly degraded (23-25). Quan- er in infected cells not previously inhibited by actinomycin titative radioautography, although cumbersome and lacking in than in uninfected cells, whether actinomycin-treated or not. the molecular specificity of physical separation technics, is a It is therefore concluded that RSV infection directly results in rather sensitive method for detecting small or transient differ- the transient synthesis of a nucleolar RNA that is not inhibited ences in intraceUular localization. by actinomycin under these conditions, whereas the concomi- RNA synthesis in the nucleolus can be selectively inhibited tant synthesis of nucleolar protein and cellular DNA is inhi- by low doses of actinomycin, probably as a result of a special bited. The possible relationship of these findings to the forma- affinity of actinomycin for guanosine- and cytosine-rich tem- tion of the mature virus particle and to the development of plate sites of precursor ribosomal RNA (21, 30, 34, 35), Pre- transformation is discussed. vious investigations have shown that a concomitantly labeled protein, assembled or synthesized in the nucleolus, is also selectively inhibited by actinomycin (34, 35). These effects, as INTRODUCTION well as the delayed inhibition of DNA synthesis (6, 19, 25, 29), are reversible by removal of the inhibitor (6, 19, 29, 42). Despite intensive investigation, the cellular pathology under- In this report, low doses of actinomycin were employed as a lying the "neoplastic" transformation of cells infected with selective inhibitor of nucleolar RNA synthesis in the expecta- tion that subsequent RSV infection might reveal discrete differences in the rates of nucleolar RNA and protein synthesis between infected and uninfected cells which in turn might elucidate some early intracellular events of infection. By 1presented in part at the Fifty-second Annual Meeting of the Federa- tion of American Societies for Experimental Biology, Atlantic City, studying the rate of recovery of nucleolar RNA synthesis from New Jersey, 1968. Federation Proceedings 27: 2801, 1968. prior inhibition with actinomycin, a significant and consistent 2present address: Laboratoire de Mddecine Expdrimentale, Coll~ge de transient increase in the labeling of the nucleolus was found in France, 11 Place Marcelin Berthelot, 75 Paris, 5 'eme, France. infected cells at a time when synthesis of nucleolar RNA re- Received September 3, 1968; accepted April 22, 1969. mained inhibited in mock-infected controls. This increase of

1598 CANCER RESEARCH VOL. 29

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1969 American Association for Cancer Research. Effect of Rous Sarcoma Virus Infection nucleolar RNA synthesis in infected cells is associated with a plied by NIH Media Section) and chased with medium con- depression of nucleolar protein and DNA synthesis, and it pre- taining a 0.1 mg/ml excess of cold lysine. After labeling, all cedes the increase of DNA synthesis accompanying cellular cells were washed five times with phosphate-buffered saline transformation (36). (Dulbecco) (10), fLxed for 30 minutes in cold 5% glutaralde- hyde (Polyscience, Inc., Rydal, Pa.), and buffered in 0.136 M sodium phosphate (18). After fixation the cells were again MATERIALS AND METHODS rinsed five times in cold distilled water, and, in the case of uridine-3H and thymidine-3H label, the acid-soluble nucleo- tides were removed by extraction with 0.5 N perchloric acid at Tissue Culture 0-4~ for 30 minutes. Secondary cultures of chicken fibroblasts were prepared using 9- to 12-day-old leukosis virus-free chick embryos (40) on Radioautography 40 x 25 mm glass coverslips in 50-mm plastic Petri dishes (Fal- con Plastics, Los Angeles, Calis UsingMedium 109 (12) supple- Radioautographs were prepared as previously described (34, mented by 8% newborn gamma globulin-free calf serum and 35) by coating the coverslip preparations with a fine-grain 10% tryptose phosphate 800,000 cells were plated; these were radioautographic liquid emulsion (Ilford L-4, diluted 1:1.6) incubated at 39.5~ in an atmosphere of 5% carbon dioxide. and exposing it at -20~ for 10-21 days. The radioauto- After 12 to 18 hours, the attached cells were washed and graphs were developed in Ficq's diaminophenol developer (13) infected. and were stained with Harris' hematoxylin. Examination with phase-contrast optics, using a Zeiss Neofluar 100/1.30 objec- tive, allowed recognition of nucleolar outlines in most cells Infection and gave adequate resolution and heightened contrast to the small silver grains. The relative areas of nucleoli and nucleus The virus inoculum used was the Bryan high-titer Rous sar- were approximately determined for each cell by multiplying coma virus strain CT-559 (7, 22) titering at 107-s focus-form- their respective largest and smallest diameters, as measured in ing units per 0.2 ml in chick fibroblast cultures (40). The cells an ocular micrometer grid. The grain counts over nucleolus, were exposed for 3 hours to a 10 -2 virus dilution (1 ml per total nucleus, and cytoplasm per cell were then determined by plate) in a diluent consisting of phosphate-buffered saline averaging 5 or more counts per cell within an approximate (Dulbecco) (10) and 2% horse serum. Estimating the plating range of 10%. The mean grain counts for samples of 25 ran- efficiency to be 10% under these conditions (40), this dose domly selected cells were thus established. Because of the was considered sufficient for the simultaneous infection of all greater consistency of the means of the ratio of nucleolar to cells. Growth medium was then replaced after a two-fold rinse. nuclear grain counts (35) than that of the means of the sepa- Uninfected control cells were treated identically except that rate grain counts, this ratio and the ratio per unit area were these cells were exposed to either the diluent alone (mock- determined for each cell. Statistical comparison of the means infected cells) or to the same virus inoculum inactivated in a was performed by variance analysis and standard "t" test, with water bath at 90~ the aid of a digital computer. The assumption of normality of Some cells were pretreated with actinomycin D (0.1 or 0.2 the sample distribution underlying a "t" test comparison of ttg/ml) for a 3-hour period and were then washed with growth the means appears justified by the fact that the differences medium prior to infection. All control cells were given identi- between the mean and the median values (Table 1) are well cal medium changes (41) and rinses to remove the inhibitor within the limits of standard error ((2o/'~/'~). and/or virus suspension. In cells labeled with tritiated thymidine, the mean incidence per mille of labeled nuclei was determined from 5 rank-listed Radioisotopes (X 1-X s) counts of 1000 cells per slide. A linear estimate of the standard deviation_ of the mean (Sx) was calculated according At varying times following actinomycin treatment and/or to the formula S X = 0.3724 (Xs-X1) + 0.1352 (X4-X2). The infection, some cells were pulse-labeled for 10 minutes with 10 estimated significance of differences between the mean inci- Bc/ml of uridine-5-3H (20 to 27 c/mmole, obtained from dence (X1, X2) was calculated from the range (w) according to Schwarz BioResearch Inc., Orangeburg, N. Y.) prepared in the formula Eagle's minimal essential medium (11). The cells to be labeled were previously rinsed with this medium in order to remove m X 1 --X 2 = t d the nucleoside from the growth medium and were "chased" 1 with growth medium containing an excess of 0.1 mg/ml of ~(Wl + w2) cold uridine. Other cells were pulse-labeled with 10 ~tc/ml thymidine methyl-3H (11.2 c/mmole) for 2 hours and chased briefly with growth medium containing an excess of 1 mg/ml RESULTS of cold thymidine, or with 10 #c/ml lysine-4,5-3H (2.9 c/mmole), obtained from New England Nuclear Corporation, Secondary cultures of leukosis virus-free chicken fibroblasts Boston, Mass.) prepared in Medium 109 without lysine (sup- were treated for 3 hours with 0.1 or 0.2/.tg/ml of actinomycin

AUGUST 1969 1599

Table 1

Time post infection Actinomycin (0.1 ~g/ml) Control: growth medium Actinomycin (0.1/~g/ml) 3 hr + growth medium 3 hr + growth medium 3 hr + RSV Growth medium 3 hr + RSV

t d = 10.82 0.10932 [0.09901] • 0.09525 0.34431 [0.35515] • 0.07722 0.08145 [0.06945] • 0.05968 0.34362 [0.35037] • 0.08746 be = 2.45 Pc = 0.014

I ta= 9.09 ta= 10.67 t a = 7.18 1 0.11246 [0.11364] • 0.09214 0.34375 [0.33334] • 0.08760 0.09055 [0.08929] • 0.07997 0.28985 [0.27995] • 0.06619

! s be = ! 4.16 t a = 7.23 t a = 5.67 t a = 2.19 P = 0.028 0.14563 [0.13910] • 0.06584 0.29869 [0.27308] • 0.09688 0.13445 [0.12821] + 0.10755 0.23793 [0.21875] + 0.07562 0.10681 [0.08131] • 0.07053 0.14895 [0.13077] + 0.08672 t a = 5.64 tae = 2.50pc = 0.012

! ! ta= 5.49 t d = 2.06 P = 0.040 0.19144 [0.17915] • 0.06804 10 0.32546 [0.31401] • 0.10184 0.30848 [0.28369] + 0.07705 0.26942 [0.26540] + 0.06005 0.18957 [0.19139];• 0.07053 0.30454 [0.28292] + 0.07618 0.25583 [0.25946] + 0.08550 t a = 4.09

! I ...... t a = 5.03 18 0.23955 [0.24020] + 0.06508 0.33405 [0.3334] + 0.06763 0.34539 [0.36148] + 0.11179 0.34323 [0.34178] + 0.07701 tde = 2.87 Pc = 0.004 I t a = 3.30 P <0.001 ] 24 0.29544 [0.27986] • 0.07652 0.29841 [0.29005]i• 0.09098 0.29953 [0.27876] + 0.05824 0.37623 [0.37279!1+ 0.10059 48 0.31011 [0.31250] + 0.08492 0.33610 [0.33112]._1+ 0.07377 0.32860 [0.31667] + 0.08681 0.36207 [0.36718][ + 0.10178 72 0.35217 [0.33517] + 0.11103 0.31757 [0.32104] 1+ 0.08080 0.34378 [0.33840] + 0.08586 0.35646 [0.3437911+ 0.09285 tae = 3.21 P = 0.0014 Effect of pretreatment with actinomycin D (0.1/~g/ml) on nucleolar RNA synthesis in Rous sarcoma virus (RSV)-infected and in mock-infected chicken flbroblast cells. One out of three representative experiments showing the rate of recovery of nucleolar RNA synthesis after inhibition by actinomycin D (0.1 pg/ml, 3 hr) in chicken fibroblast cells subsequently infected with Rous sarcoma virus or mock-infected with a 10-min pulse of uridine-3H. Mean [Median] (n = 25) of the ratio per cell of the grain count over the nucleolus/grain count over the total nucleus + standard deviation (o) Mean 1 -- Mean 2 ta= 2 2 (01 Inl) + (o 21n2)

tdc= t a relative to the control (column 2) P, (Pc) = significance of difference of the means expressed as probability of random error Duplicate values represent separate samples from either the same or duplicate culture dishes. The t a values of adjacent means are between the dividing vertical lines. The t a values of nonadjacent means are on the outside of the respective indicator bracket. This is the same experiment as Charts 1 and 2.

and were washed and infected with Bryan strain RSV as de- (per cell) of the nucleolar to total nuclear grain count (Table 1 scribed above. Control cells were either mock-infected in the column 2; also see Ref. 36). suspending medium or exposed to a heat-inactivated virus sus- The effect of pretreatment with actinomycin on nucleolar pension (Chart 3). At various times following infection, the cells RNA synthesis in infected and uninfected cells was compared were pulse-labeled with tritiated uridine for 10 minutes so that in three experiments (Table 1; Charts 1-3, 5). In mock-infec- the label would appear only in the nucleus. Mean grain counts ted cells there is a significant selective inhibition of nucleolar of the nucleolus and of the nonnucleolar areas of the nucleus RNA synthesis in cells pretreated with actinomycin, but this were determined through radioautographs using groups of 25 inhibition is gradually reversed within 24 hours after removal randomly selected cells; statistical comparison was made by of the inhibitor. However, in cells infected with RSV after standard "t" test analysis (Table 1). Although the standard actinomycin treatment, the inhibition is already reversed with- deviation for the absolute nucleolar grain counts is on the in 10 hours (Table 1, Chart 1). At that time nucleolar RNA average of 52.4% of the mean, it is reduced to 26.1% (o/x/~= synthesis in actinomycin-treated, RSV-infected cells shows a 5.2%) when the nucleolar grain count is expressed as the ratio highly significant increase, above that of similarly treated but

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1A 1B W-~ dO RSV RSV AD 1 Zz 71 200 _jb_ A.. O0 / \'. W

oZ ...... ~W ,,j,+- - ~..___ _...... ,,_ 100 I.j_ t.l-I '~,4./. -~ ...... -~" '" """ -o o n 70 F_03 7< o 50 L) n~ a-..-~ADShr +RSV:ADShr +GM z < n [] AD 5hr § RSV CONTROL 9--'--- AD 5 hr + RSV'RSV 50 n~z o.--.-o AD 5 hr + GM" CONTROL v vRSV'CONTROL Lg~

7c~ l I I I I I I I I I I I I l

W 0 12 24 48 72 0 12 24 48 72 HOURS POSTINFECTION

Chart 1. (Same experiment as Table 1 and Chart 2.) Effect of actinomycin D (0.1 #g/ml for 3 hours) on nucleolar RNA synthesis per unit area in Rous sarcoma virus (RSV)-infected cells and in uninfected chicken fibroblast cells (uridine-3H, 10 min pulse label). Changes with time after infection of the mean grain count per unit area of nucleolus/nucleus expressed as percentage of the respective controls. GM, growth medium. 1/1, zx...... A, cells treated with actinomycin D (AD) and subsequently infected with RSV are compared with cells pretreated with AD and subsequently mock-infected, o--u, cells treated with AD and subsequently infected are compared with control cells, i.e., changes in growth medium instead of AD and mock-infected, o--o cells treated with AD and subsequently mock-infected are compared with control cells. 1B, 9...... 9 cells treated with AD and subsequently infected are compared with infected cells not treated with AD. v--v, infected cells not treated with AD are compared with control cells.

uninfected cells (Table 1). This is shown both as an increase in

AUGUST 1969 1601

Groin Count Nucleolus :t:~-- dissociated briefly by actinomycln from ribosomal ribonu- 1 8.76 13.85 8.97 25.96 cleoprotein in the nucleolus. :1:2.79 t- 1.59 + .91 + 2.79 As shown in Chart 5, there is an identical 50% reduction of Nucleus DNA-labeled nuclei in infected and uninfected ceils 10-18 51.58 45.87 59.53 71.99 hours after removal of actinomycin as well as a decrease in the IOO- I-- + 7.23 1.5.05 +5.75 +7.93 relative density of the label per cell at 18 hours. 3 However, Z o Do ~. Nucleolus % during this period, nucleolar RNA synthesis of actinomycin- o Nucleus treated infected cells is increased twofold (Chart 5). This inhi- z 4c- bitory effect of actinomycin on DNA synthesis in both infec- n.- --T- 0 ted and uninfected cells is reversed within 24 hours. RSV-

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RSV 5A inhibited. This increase of RNA synthesis in the nucleolus of a 300, actinomycin-treated, infected cells was observed 10 to 18 ILl -~D1 Uridine-3Hgraincount in Z ./ Nucleolus/Nuc/eusAD 3hr +RSV. hours following infection and preceded a comparable relative AD 3 hr § Growth Medium increase in RSV-infected control cells by several hours. >_n"~;o 200 It may be concluded from these s that: (a) the ob- i , i - ...~ served increase of nucleolar RNA label is a result of infection, LUg) OU. (b) the nucleolus in infected cells is transiently a site of assem- ~o bly of rapidly labeled RNA with a different rate of synthesis '-z 100 --N~.._.-=--__~='.~__.~-~_ i .... than in control cells, and (c) this increase of nucleolar RNA ~L~ synthesis is not inhibited by actinomycin in infected cells Z~ ii 5B '~UJ under these conditions. It is possible that this increment of nucleolar RNA label, not inhibited by low doses of actino- -'~"~/ MEAN INCIDENCE OF THYMIDINE-3H - mycin in infected cells, represents a viral RNA, An alternate n~u.i- 50 LABELED NUCLEI PER THOUSAND n=5 t~L) possibility is that cellular ribosomal RNA synthesis is stimu- 0.D 40 n o AD (0.2/zg/rnl) z - :5 hr + RSV : Control lated as a consequence of infection. Such a transient increase w o----o AD (0.2p.g/ml) of nucleolar RNA synthesis could resuk from competition be- hlJz,,, 30 - 3hr +GM:Control e~UJ tween a viral RNA and actinomycin for guanosine- and cyto- z~--.--~ AD (0.2p.g/ml) sine-rich template sites of ribosomal precursor RNA in the :Shr +RSV:AD3hr +GM nudeolus (21, 30). The ultrastructural localization of this RNA and actinomycin (31) in the nucleolus of infected cells may I I I I I I I distinguish between these interpretations. 0 12 24 48 72 TIME POSTINFECTION RSV (hours) The increase of nucleolar RNA synthesis in infected cells previously inhibited by actinomycin is not accompanied by a Chart 5. (Same experiment as Chart 6). Inhibition and recovery of corresponding increase in nucleolar protein, but, in both in- DNA synthesis (and nucleolar RNA synthesis) after actinomycin fected and uninfected cells, nucleolar protein label is inhibited treatment (0.2 #g/ml) in infected and mock-infected cells expressed as a identically by actinomycin. In other cell systems, such protein percentage of the respective controls. 5,4, a...a, Mean grain count over nucleoli (n = 25) after uddine-3H (10-min pulse label) as ratio of total RNA and expressed as percentage of control, i.e., cells treated with actinomycin (0.2 #g) and subsequently infected compared with cells • RSV 6A It) pretreated with actinomycin but subsequently mock-infected. 5B, Mean I incidence of labeled nuclei per thousand (n = 5) after 2-hour pulse label hi Z of Thymidine-3H GM, growth medium, omn, in ceils treated with F, j 300 actinomycin D CAD) and subsequently infected (expressed as ~g percentage of control), o..... o, in cells treated with AD and

subsequently mock-infected (expressed as percentage of control). ~tL o 200 zx...... A, in ceils treated with AD and subsequently infected expressed as Oh percentage of AD treated cells subsequently mock-infected (same curve /- in Chart 6). ~WC <~ 100

-1- 70 "\ ..... / i~...... "~...... -all DISCUSSION %.. "...... ~....~.., I1= (.) "... / w~ The radioautographic experiments reported here were de- o-z 50 signed to investigate the functions of the nucleohs with re- z_J spect to synthesis or assembly of RNA and protein in early v v RSV" Control am stages of RSV infection (8, 15, 16; S. W. C. Fiske and F. z~--.--~ADShr +RSV:AD3hr +GM Haguenau, unpublished data). When the rate of recovery of e ...... m'AD3hr +RSV:RSV nucleolar RNA synthesis from selective inhibition by low z I ,1 I I I I I w doses of actinomycin (34, 35) is compared in infected and 0 12 24 48 72 uninfected cells, significant differences in RNA labeling of the TIME POSTINFECTION RSV (hours) nucleolus were observed. It has been shown that the rate of synthesis of rapidly labeled RNA in the nucleolus of cells in- Chart 6. (same experiment as Chart 5; see legend of Chart 5B) Effect fected with RSV after actinomycin inhibition is transiently of Rous sarcoma virus (RSV) infection on the rate of increase of DNA synthesis. Mean incidence of labded nuclei per thousand (n -- 5) after increased, both absolutely and per unit area, above the level 2-hour pulse label of Thymidine-3H v. v, RSV-infected cells observed in actinomycin-treated cells mock-infected or ex- expressed as percentage of mock-infected control cells. =.... a, posed to inactivated virus. Also the proportion of total RNA Actinomycin D (AD)-treated cells subsequently infected expressed as synthesized in the nucleolus of these infected cells is greater percentage of infected cells not treated with AD. zx...... A same curve per unit area than in uninfected control cells not previously as Chart 5.

AUGUST 1969 1603

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1969 American Association for Cancer Research. R. Gerald Susla'ncl, Thomas W. Pry, and Giancarlo F. Rabotti label is associated with ribosomal precursor RNA in nucleoli their Control. J. Natl. Cancer Inst., 16: 287-311, 1955. (21, 27, 44, 45) and is similarly inhibited selectively by low 8. Bukrinskaya, A. G., Burducea, O., and Vorkunova, G. K. The Intra- doses of actinomycin (34, 35). These observations suggest the cellular Site of RNA Synthesis of Newcastle Disease Virus. Proc. possibility that actinomycin may briefly dissociate nucleolar Soc. Exptl. Biol. Med., I23: 236-238, 1966. RNA from ribosomal ribonucleoprotein in infected cells. Such 9. CampbeU, J. B., Maes, R. F., Wiktor, T. J., and Koprowski, H. The Inhibition of Rabies Virus by Arabinosyl Cytosine. Studies on the a host protein may be required for the assembly of intraceUu- Mechanism and Specificity of Action. Virology, 34: 701-708, lar viral RNA or its transport to the virus particle (23). If 1968. inhibition of synthesis of such a nuchoprotein results in acti- 10. Dulbecco, R., and Vogt, M. Plaque Formation and Isolation of vation of additional template sites for either ribosomal (1) or Pure Lines with Poliomyelitis Viruses. J. Exptl. Med., 99: viral RNA synthesis, the paradoxic increase of nudeolar RNA 167-182, 1954. label in actinomycin-treated infected cells above that of in- 11. Eagle, H. Amino Acid Metabolism in Mammalian Cell Cultures. fected control cells (here reported) may be explained. A simi- Science, 130: 432-437, 1959. lar mechanism, possibly related to interferon induction, has 12. Evans, V. J., Bryant, J. C., Fioramonti, M. C., McQuilkin, W. T., been demonstrated in other cell-virus systems (8, 9). Sanford, K. K., and Earle, W. R. Studies of Nutrient Media for Tissue Culture CeLls In Vitro. Cancer Res., 16: 77--94, 1956. It has also been shown that DNA synthesis is inhibited iden- 13. Ficq, A. Autoradiography. In: J. Brachet and A. E. Mirsky (eds.), tically in both infected and uninfected cells by actinomycin The Cell, pp. 1-77. New York: Academic Press, Inc., 1950. (2-5, 20, 38, 39, 42, 43) at a time when nucleolar RNA 14. Goldd, A., and Vigier, P. Kinetic Study of the Inhibition of Growth synthesis is increased in infected cells. of Rous Sarcoma Virus and Cells by 5-Fluorouracil In Vitro. Virol- Subsequent differences in the rate of increase of DNA ogy, 20: 420-432, 1963. synthesis between infected control cells (17, 28, 36) and in- 15. Haguenau, F., and Beard, J. W. The Avian Sarcoma-Leukosis Com- fected ceils pretreated with actinomycin may be related to the plex; Its Biology and Ultrastructure. In: A. J. Dalton and F. well-known inhibitory effect of actinomycin on cellular trans- Haguenau (eds.), Tumors Induced by Viruses: Ultrastructural formation (4, 38, 42) and would suggest that this effect may Studies, Vol. 1, pp. 1-59. New York: Academic Press, Inc., 1962. 16. Hampton, E. G., and Eidinoff, M. L. Transformation of Chick be a secondary result of inhibition of cellular DNA synthesis Embryo Cells in Culture by Rous Sarcoma Virus-Cytochemical by actinomycin at a critical period of genotypic FLxation (20). Studies. Cancer Res., 22: 1061--1072, 1962. However, a nucleolar RNA produced as a consequence of in- 17. Macieira-Coelho, A., and Pontdn, J. Induction of the Division fection is not inhibited by actinomycin under these condi- Cycle in Resting Stage Human Fibroblasts after RSV Infection. tions. Biochem. Biophys. Res. Commun., 29: 316--321, 1967. 18. Millonig, G. Further Observations on a Phosphate Buffer for ACKNOWLEDGMENTS Osmium Solutions. In: S. S. Breese, Jr. (ed.), Electron Microscopy: Fifth International Congress on Electron Microscopy, Philadelphia, The authors wish to express their appreciation to Mr. George Shakarji Pa., August 29, 1962, P-8. New York: Academic Press, Inc., of the Division of Computer Research and Technology, NIH, for pro- 1962. gramming the raw data, to Dr. R. Luginbuhl, Department of Agri- 19. Mueller, G. C., and Kajiwara, K. Actinomycin D and p-Fluoro- culture, University of Connecticut, Storrs, Connecticut, and to the NIH phenylalanine, Inhibitors of Nuclear Replication in HeLa Cells. for supplying the RIF-free chick embryos, and to Dr. Elmar Alpert, Biochim. Biophys. Acta, I19: 557--565, 1966. Merck, Sharp and Dohme Research Division, West Point, Pennsylvania, 20. Nakata, Y., and Bader, J. P. Studies on the Fixation and Develop- for his generous gift of actinomycin D. ment of Cellular Transformation by Rous Sarcoma Virus. Virology, 36: 401-410, 1968. 21. Perry, R. P. On Ribosome Biogenesis. In: International Symposium REFERENCES on the Nucleolus, its Structure and Function. National Cancer In- stitute Monograph No. 23. Washington, D. C.: U. S. Government 1. Allfrey, V. G., and Mirsky, A. W. Role of Histone in Nuclear Printing Office, 1966. Function. In: J. Bonnet and P. T~o (eds.), The Nucleohistones, 22. Rabotti, G. F. Oncogenic Effects of Rous Sarcoma Virus Strain pp. 267-288. San Francisco: Holden-Day, Inc., 1964. CT-559 in Dogs. In: Recent Developments in Comparative Medi- 2. Bader, J. P. The Role of Deoxyribonucleic Acid in the Synthesis of cine, Zoological Society of London, pp. 363--370. New York: Aca- Rous Sarcoma Virus. Virology, 22: 462--468, 1964. demic Press, Inc., 1966. 3. Bader, J. P. The Requirement for DNA Synthesis in the Growth of 23. Robinson, W. S. The Nucleic Acid of Rous Sarcoma Virus. In: W. Rous Sarcoma and Rous, associated Viruses. Virology, 26: 253- J. Burdette (ed.), Viruses Inducing Cancer, pp. 107-124. Salt Lake 261, 1965. City: University of Utah Press, 1966. 4. Bader, J. P. Metabolic Requirements for Infection by Rous Sar- 24. Robinson, W. S., and Baluda, M. A. The Nucleic Acid from Avian coma Virus. I. The Transient Requirement for DNA Synthesis. Myeloblastosis Virus Compared with the RNA from the Bryan Virology, 29: 444-451,1966. Strain of Rous Sarcoma Virus. Proc. Natl. Acad. Sci. U. S., 54: 5. Bader, J. P. Metabolic Requirements for Infection by Rous Sar- 1686-1692, 1965. coma Virus. II. The Participation of Cellular DNA. Virology, 29: 25. Robinson, W. S., Pitkanen, A., and Rubin, H. The Nucleic Acid of 452-461, 1966. the Bryan Strain Rous Sarcoma Virus: Purification of the Virus 6. Baserga, R., Estensen, R. D., Petersen, R. O., and Layde, J. P. Particle and Isolation of the Nucleic Acid. Proc. Natl. Acad. Sci. Inhibition of DNA Synthesis in Ehrlich Ascites Cells by Actinomy- U. S., 54: 137-144, 1965. cin D. II. The Presynthetic Block in the Cell Cycle. Proc. Natl. 26. Robinson, W. 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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1969 American Association for Cancer Research. Effect of Rous Sarcoma Virus Infection on Recovery of Nucleolar RNA, Ribonucleoprotein, and DNA Synthesis from Inhibition by Actinomycin D

R. Gerald Suskind, Thomas W. Pry and Giancarlo F. Rabotti

Cancer Res 1969;29:1598-1605.

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