[CANCER1RESEARCH36 Part 1, 1839-1846,September 1966]

Effects of Actinomycin D on Avian Myeloblast and BAI Strain AVirus RNA Synthesis in Vitro1

ROSEMARIE ZISCHKA, A. J. LANGLOIS, P. R. RAO, R. A. BONAR, AND J. W. BEARD

Department of Surgery, Duke University Medical Center, Durham, North Carolina

Summary

Avian myeloblasts infected with BAI strain A avian tumorvirus were cultured in vitro and treated with actinomycin D.The effects of the drug on cell growth and death and the synthesis of cell and virus RNA were examined. Actinomycin D (AD)concentrations of 0.01-0.05 tig/ml caused cell damage and death

at proportional rates, and, as shown in earlier work, inhibited cellRNA synthesis up to 60% without depressing virus RNA synthesis or virus particle liberation. In comparison, 0.5 fig AD/mlcaused more rapid cell death with parallel inhibition of 70-85%

of cell RNA synthesis and 70% of virus RNA synthesis but didnot decrease the output of particles having the characteristicvirus adenosinetriphosphatase activity. These results are discussed and compared with findings with other virus-host systems.

Introduction

Assembly and elaboration of the BAI strain A avian tumorvirus (7) occurs by particle budding at the external cell membrane (39) of myeloblasts from birds with myeloblastic leukemia(15) induced by the agent. Evidence of other aspects of cellinvolvement in the synthesis and formation of the agent containing RNA, lipid, and protein (12) has been sparse. Tracerstudies (35) indicated that synthesis of viral RNA involved poly-nucleotides occurring in the soluble fraction of the cells.

Results with other virus-cell systems suggested that AD2,which binds reversibly to DNA (14, 17) and inhibits DNA-dependent RNA synthesis (29) might be used to investigatethe possible role of DNA in the synthesis of BAI strain A virusRNA by myeloblasts. Formation of some RNA viruses such asMengo (16, 27, 28), polio (33, 44), and vesicular stomatitis (2),has been reported to proceed in the presence of AD Tobaccomosaic virus RNA is synthesized (31), although there may bea small reduction in infectious agent yield in the presence of theantibiotic. Results with Newcastle disease agent have variedwith experimental conditions (6, 21, 24, 30, 43) from enhancement of virus synthesis to about 90% inhibition of the process.Elaboration of infectious influenza (6, 21) and fowl plague (4, 5,

1This work was aided by a research grant (C-4572) to DukeUniversity from the National Cancer Institute, NIH, USPHS;by a grant from the American Cancer Society, Inc. (E-84A); andby the Dorothy Beard Research Fund.

2The abbreviations used are: AD, actinomycin D; ATPase,adenosinetriphosphatase; RSV, Rous sarcoma virus.

Received February 7, 1960.

30) viruses was inhibited more than that of Newcastle diseasevirus, but the results were strongly influenced by experimentalconditions. Influenza virus-induced RNA synthesis was diminished, although it was less sensitive to the drug than was virusproduction. In another study (13) with a different strain of influenza and with fowl plague virus, no inhibition of virus-inducedRNA synthesis was seen. Differences in antibiotic dose, celltype, and methods of assay all influence the results of the inhibitor studies and make a close comparison of the various results with the myxoviruses difficult. Also of interest was thefinding (19) that AD could inhibit the in vitro synthesis of RNAusing reovirus RNA as template and an RNA polymerase fromEscherichia coli. Reovirus formation appears to be enhanced atlow concentrations of AD and inhibited by higher levels of theantibiotic (20, 34).

Closely related to the present work were findings with 2 aviantumor virus-host cell systems. AD sharply depressed synthesisof Rous sarcoma virus RNA (37) and of infectious Rous sarcomavirus (2, 3, 36, 40, 41) and "Rous-associated virus" (3) when

added to cultures of chick embryo cells close to the time of infection. Viras elaboration by established Rous cells was muchless sensitive (36, 40, 41) to the drug, and the inhibition was reversed on removal of AD. It has been reported briefly (1) thatsynthesis of BAI strain A virus RNA by infected chick embryofibroblasts was inhibited by very low concentrations of AD addedat the time of infection.

In the present work, we investigated the effects of AD ongrowth of and RNA synthesis by avian leukemic myeloblasts andon elaboration of BAI strain A virus and vims RNA synthesisby the myeloblasts cultured in vitro. As already reported (45),addition of AD to myeloblast cultures resulted in marked declinein cell RNA synthesis, while, at the concentrations used then,the rate of virus RNA synthesis did not decrease. Work withhigher concentrations of AD has shown inhibition of viral RNAsynthesis as well as rapid cell death. The present work includedstudies on virus liberation into culture fluids in relation to cellgrowth and damage. Morphologic cell changes were examinedby light and electron microscopy. The biochemical and viruselaboration studies are considered in this report, and the ultra-structural features of AD-treated myeloblasts will be describedseparately (22).

Materials and Methods

MYELOBLASTCULTURES.White Leghorn chicks of Line 15(Regional Poultry Research Laboratory, East Lansing, Mich.)(42) were inoculated with BAI strain A virus at 3-5 days of

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Rosemarie Zischka, A. J. Langlois, P. R. Rao, R. A. Bonar, and J. W. Beard

age. Birds developing high concentrations of leukemic myelo-blasts in the peripheral circulation determined by daily bloodsmears and high content of virus in the plasma estimated byadenosinetriphosphatase (ATPase) activity (11)—18-32 daysafter inoculation—were bled by heart puncture. Myeloblastscollected by centrifugation of the heparinized blood were washedin Gey's saline solution and suspended for culture by the proce

dures previously described (11) in a mixture of equal parts ofchicken serum and Medium 199 with penicillin, streptomycin,and added folie acid. All cultures were incubated at 37°Con a

rotatory oscillator.In some experiments, cell growth and virus release were studied

in the usual (11) 5-ml cultures containing 2.5-5 X IO7cells/mlincubated in 50-ml Erlenmeyer flasks. Medium was changed andcultures were divided at appropriate intervals (11), 2-3 days ingrowing cultures. When larger amounts of material were required, similar cultures were of 30- or 60-ml volumes in 500-mlflasks. Cultures containing about IO8cells/ml were used in someshort-term studies. Fresh cultures were maintained for 7-15 daysbefore study to permit attainment of the "equilibrium" state

(10) of exponential cell growth and constant rate of virus liberation. Attributes of cell growth and virus formation vary (11)with cells from different donors, and cultures were selected forsuitable behavior in these respects. Viable cells were counted ina hemocytometer by distinction from "nonviable" elements

staining with trypan blue (10). Numbers of virus particles were

_ 1000"0

g 100-f

Ê

E IO

IOO--

IO-'

AD =0.001 /iq/ml

AD = 0.

t

AD= 0.01

-100

-10

\

A0 = O.

1000

--IOO

-io Qs

O 5 IO 15 20 25 30 O 5 IO 15 20 25 30

DAYS OF CULTURE

CHAUT1. Cell growth and virus release in 5-ml myeloblast cultures containing different concentrations of AD. X X indicates virus particle number; and O and •represent viable cellcounts. The solid line was fitted (10) on the assumption of logarithmic cell growth, and the dash line on the assumption that the rateof virus particle release was constant/cell/hr. All values werethe average of estimates on triplicate cultures. The arrows indicate the first cell change to medium containing AD, and 0's

indicate change to fresh medium containing the antibiotic. Cellconcentration was maintained ¡it2-5 X 107/ml.

TABLE 1EFFECTS OF ACTINOMYCIND ONMYELOBLASTGROWTHANDVIRUS

RELEASE IN 5-ml SHORT-TERM CULTURES

ACTINO-HYCINDNone0.10.20.5EXPOSURE

TO ACTINOITÕCINDViable

cells(/ml

XIO-7)0

hr9.710.110.910.44hr10.710.812.210.624hr12.211.27.73.2(%

of totalcells)0

hr939696984hr9694928624hr91775626Virus

particles/ml X 10-'°(24hr)ATPase3.73.54.44.9Direct

count3.53.64.64.3

estimated by ATPase activity measured in reference to a standard of virus content determined by electron microscopic particleenumeration (32). Virus amount was measured in some studiesby both enzyme activity and direct particle count. In some experiments, quantitative relationships between cell growth andvirus release were analyzed as previously described (10).

Treatment with AD (obtained from Merck, Sharp and Dohme,West Point, Pa.) was effected by diluting fresh aqueous antibiotic solution in desired concentrations in whole culture medium. The number of viable cells in a culture was determined,and the cells recovered by centrifugation were resuspended inthe medium containing AD. Cell exposure to antibiotic was continuous in the periods of study, and necessary changes of culturefluid were made with medium containing AD in order to providefor the additional DNA formed in the cultures still growing inlow AD concentrations.

Incorporation of tritium-labeled uridine, specific radioactivity3 c/mmole (New P^ngland Nuclear Corporation, Boston, Mass.),was studied by addition of 1 or 5 /uc of uridine-3H/ml of culture.

RNA EXTRACTION.For studies on cell RNA synthesis, myelo-blasts were washed 3 times with Gey's saline solution after exposure to uridine-3H and broken with a Wig-L-Bug (CrescentDental Manufacturing Co., Chicago, 111.)for 1 min using the 2-ml vial and 2 ball pestles. All cells were broken in this period, andthe results were uniformly reproducible with small amounts ofmaterial. The nucleic acids were extracted with hot 10% NaCland precipitated with ethyl alcohol, and RNA was separated fromDNA by alkaline hydrolysis as described (35). The RNA wasdetermined by absorption at 260 mju. The ultraviolet absorptionspectrums indicated a high degree of purity. RNA solutions wereevaporated to dryness, and the residues were transferred to vialsfor radioactivity measurement in a liquid scintillation counter(Packard Instruments Company, Inc., La Grange, 111.)as described (35).

VIRUSISOLATION.Virus was purified by 4 cycles of alternatelow and high speed centrifugation (12) of culture fluids. Afterthe last centrifugation, virus was resuspended in 0.2 ml water plus1 ml Hyamine (10 X) and transferred to counting vials for radioactivity determination. In the earlier part of these studies, someof the virus suspensions were treated with ribonuclease and colduridine as described before (35) to test for possible contaminationby cell RNA. No difference was found between treated and untreated preparations, and the treatment was omitted in later

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Aclinomycin D and RXA Synthesis

24CONTROL ACTINOMYCIN 0

24

20

,6 ÃŽ'b

12

LJO

12 15 18 21 24 03

HOURS

9 12 15 18 21 24

GHAUT2. Cell growth and death and virus release over 24 hr in myeloblast cultures with and without 0.5 fig Al)/ml added at 0 time.The lines were fitted (10) to the total cell count by assuming logarithmic cell growth for 18 hr and to the virus particle count by assuming constant rates of virus production/cell (total)/hr for 18 hr. Dashed portions of the lines indicate extrapolations.

2.5-5 X IO7myeloblasts/ml, the cells grow exponentially for in

definite periods and liberate virus into the culture fluid at constant rates/cell/hr (11). The agent released can be estimatedquantitatively in terms of numbers of physical virus particles byATPase assay standardized by electron microscopic particleenumeration, as already shown (9) in earlier tissue culture experiments. Detection of departures from the usual pattern of cellgrowth and virus release can be facilitated by mathematical description of the data (10).

Chart 1 shows that continued cell exposure to 0.001 jugAD/mlin such cultures did not influence either cell growth or rate of virus release. The small transient decrease in rate of virus output inthis experiment was not significantly different from that frequently observed in myeloblast cultures (11). In contrast, higherantibiotic concentrations caused cell damage and death afterdecreasing periods of exposure, and with 0.1 /ig/ml, essentiallyall of the cells were nonviable within 48 hr.

It is notable, however, that virus output, as indicated by thismethod of analysis, did not decline appreciably with onset of decline in the number of viable cells. This was particularly noticeable in the experiment with 0.05 /ngof antibiotic in Chart 1.

For determination of effects on cell and virus RXA synthesis,it was desirable to employ antibiotic concentrations sufficient toexert specific influence without death of the cells within the periodof biochemical study. Since 0.05 jug AD/ml (Chart 1) had no inhibitory effect on virus RXA synthesis (45), the time course ofeffects of higher concentrations was examined. Table 1 gives thedata obtained in a 24-hr experiment to determine the effects ofantibiotic concentrations of 0.1, 0.2, and 0.5 /ng/ml on cell growthand viability and virus output. Single control and treated 5-mlequilibrium cultures but containing about IO8 myeloblasts/ml

in this experiment were used for each concentration. Little effect

0.01 0.05 O.I 5.0

ACTINOMYCIND (¿ig/ml)

CHAUT3. Effect of different concentrations of Al) on incorporation of uridine-3!! into myeloblast cell UNA. Cells at concentrations of 3-5 X 107/ml were exposed to AD for 3 hr, then uridine-'Hwas added for 3 or 6 hr, and the UNA was extracted.

virus preparations. Recovered virus was determined by ATPaseactivity of the agent obtained in the 3rd sedimentation cycle.

CELLEXAMINATION.Cells from various cultures were examinedby phase contrast microscopy, and samples were taken for ultra-structural study described in another report (22).

Results

Preliminary studies were made to determine the influence ofdifferent concentrations of AD on myeloblasts as usually maintained in vitro. Under the conditions of 5-ml volumes containing

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Rosemarie Zischka, A. J. Langlois, P. R. Rao, R. A. Bonar, and J. W. Beard

TABLE 2EFFECT OF ACTINOMYCIND ON TJRIDINE-3H UPTAKE INTO RNA OF CELLS AND VIRUS

IN AVIAN MYELOBLASTOSISEach value is the mean of 3 separate flasks. Values for inhibition of viral RNA synthesis were calcu

lated from total radioactivity to avoid the errors in estimating the small numbers of virus particlesproduced.

5-hrexperiment"Total

radioactivityCellUNA(mg)(cpm)Specific

radioactivity (cpm/jugRNA)Inhibition(%)10-hr

experiment*Total

radioactivityCellRNA(mg)(cpm)Specific

radioactivity (cpm//igRNA)Inhibition(%)VIRUSNo

actinomycin5410831Actinomycin(0.5»ig/ml)20906120376CELL

RXANo

actinomycin37.1

X10s7.847516.7

X10'3.0556Actinomycin

(0.5„g/ml)8.57

XIO56.2138715.44

X10"2.918866

»For 2 hr, 9 X 10' cells/ml were exposed to 0.5 ¿igactinomycin D/ml, and uridine-3H (5 MC/ml) was

then added for 3 hr.6 For 3 hr, 1.4 X 10s cells/ml were exposed to 0.5 /ng actinomycin D/ml, and uridine-3!! (5/zc/ml) was

then added for 7 hr.

was exerted in 4 hr by 0.1 ;ug AD/ml, but approximately 23% ofthe cells in this preparation were nonviable after 24 hr. Largeramounts of antibiotic caused more rapid cell death. With 0.5 /xg,14% of the cells were nonviable in 4 hr, and 74% were nonviableafter 24 hr. Evidence of cell nonviability obtained by trypanblue staining was closely paralleled by clumping of chromatinand vacuolization of the cytoplasm observed by phase contrastand electron microscopy (22). Other like studies revealed moderate variations in the behavior of cells from different chickdonors. In this study, determination of virus particle liberationby ATPase assay was checked by particle counts in the electronmicroscope. The results of the 2 methods (Table 1) agreed withinexperimental error, and particle formation appeared to be somewhat higher with AD treatment.

In order to determine more clearly the course of cell growth anddeath and virus release, a more detailed study was made by cellcounts and virus estimate at brief intervals in short term cultures.For this purpose 50-ml cultures containing about 10s cells/ml

were prepared for treatment with 0.5 /ug AD/ml and for parallelcontrol studies without the antibiotic. Samples were taken at3-hr intervals. The high concentration of cells was used in order toobtain amounts of virus sufficient for ATPase measurements.Chart 2 shows the data for total and viable cells and for viruselaboration.

The lines were fitted to the 0- to 18-hr data by a least squares

method (10) based on the total cell population (instead of theusual viable cell base) and on the assumption that the rate of cellincrease was logarithmic and that virus production/cell/hr wasconstant. The fit of the lines to the experimental points is reasonably good and appears to support the 2 assumptions. The controland AD-treated cultures had generation times of 2.8 and 2.1 days

and rates of virus elaboration of 21 and 22 virus particles/cell/hr,respectively. These values are in the normal range for myeloblast

cell cultures. The proportion of nonviable cells in the control culture remained at approximately the 5% level, and the total number of cells in the control thus reflected principally the number remaining viable. It is probable that decline in the cell growth inthe control culture after 18 hr was related to the high concentration of myeloblasts and consequent depletion of the culture medium.

In contrast, increase in nonviable cells in the treated culturebegan at about 6 hr, and the number of viable cells declined progressively. At the end of 24 hr, the numbers of viable and non-

viable cells were approximately equal. That cell growth continueddespite the lethal action of antibiotic on some myeloblasts, however, was shown by the continued increase in total cells in thetreated culture until the 21-hr period.

An impressive feature of the results was the apparently continued elaboration of virus particles, as measured by ATPaseactivity in cultures containing a high proportion of cells whichwere nonviable as judged by staining with trypan blue (Charts1 and 2) and which showed marked morphologic change (22). Todetermine whether the ATPase activity was associated withparticipate material, supernatant fluids from control and AD-

treated cultures were centrifugée!at 3000 X g for 15 min and30,000 X g for 45 min, and the ATPase activity in pellets andsupernatant« was determined. The sedimentation behavior ofthe enzyme activity was that expected of BAI strain A virus influid from both treated and untreated cultures; activity wasfound almost entirely in the supernatant fluid after low-speed

centrifugation or in the pellet obtained with the higher speed.Myeloblasts exhibit ATPase activity at the cell surface (39) and,consequently, there was the possibility that the ATPase activityin the culture fluid might have been due to membrane fragmentsof disintegrating cells. That this was unlikely in the first 18 hr ofthe experiment is indicated in Chart 2 by the close correspondence

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Actinomycin D and RNA Synthesis

in

IO O 2 4 6 8 IO

HOURS

CHART4. Cell growth and death and virus formation measured hourly in control cultures and in cultures from the same stock treatedwith 0.5 MgAD/ml at 0 time. Uridine-'H was added at 3 hr, and cells and virus were collected at 10 hr, extracted, and analyzed to provide the data on RNA synthesis shown in Table 2. Each point represents the mean of 3 culture flasks. The lines were fitted as in Chart 2with use of the 0-10 hr values for the control and the 0-6 hr values for the AD-treated culture.

lar or partially formed virus particles coincidental with cellbreakdown. Such increase in ATPase activity corresponding tothe time of cell breakdown and decrease in number of total cellswas observed, also, in other experiments (Chart 4).

AD EFFECT ON MYELOBLAST CELL AND VIRUS RNA SYNTHESIS.

The effects of various concentrations of AD on the uptake ofuridine-3H into myeloblast cell RNA are shown in Chart 3 for cellconcentrations of 3-5 X 107/ml. The degree of inhibition with agiven level of AD was somewhat less with higher cell concentrations reflecting the larger amount of DNA available to bind thedrug. With 0.5 jugAD and 1 X IO8cells/ml, for example, uptakeof uridine-3H into cell RNA was inhibited about 70% (Table 2)

as compared with 86% inhibition in cells cultured at the lowercell concentration.

In an earlier study (45) it was found that low concentrations ofAD (0.01 and 0.05 jug/ml) did not inhibit uptake of uridine-'Hinto viral RNA even though a concentration of 0.05 jug/ml wassufficient to cause about 60% inhibition of cell RNA synthesis(45) as well as characteristic nucleolar reorganization (22) and,in time, cell death (Chart 1). It was desirable to extend the studies to higher concentration of AD, if it was possible to do so withintact cells. The results shown in Table 1 and Chart 2 indicatedthat the effect of 0.5 ¿tgAD/ml on RNA synthesis could bestudied for time periods of approximately 6 hr without extensivecell death and degeneration.

The results of 2 experiments with 0.5 jug AD/ml in culturescontaining about 10* cells/ml are given in Charts 4 and 5 andTable 2. In the experiment of Chart 4, progressive decrease in thenumber of viable cells began at 6 hr. In the total period of 10 ruin this experiment, the uridine-3H uptake measured in the interval of 3-10 hr exposure to AD indicated inhibition of both celland virus RNA synthesis (Table 2).

In order to restrict the observations only to viable cells, thestudy of Chart 5 was limited to a 5-hr period during which therewas no increase in nonviable mveloblasts. Nevertheless, in this

IO1"i

8ooto

6LUo1—

tu4o.in

ü25i(ji^\/iable

Cells}f^-~T*S^-^J^*~~^>e^=^—ControlActinomycin

D-Virus

x^.^iC^5^""**"

Non vio bleCellsDI

234!IOe6C42'oHOURS

CHART5. Viable and nonviable cells and virus particles in control cultures and in cultures treated with 0.5 pg AD/ml at O hr.Uridine-'H was added at 2 hr, and cells and virus were collectedat 5 hr and extracted and analyzed to provide the data on uri-dine-'H incorporation into RNA shown in Table 2. Each pointrepresents the mean value of 3 culture flasks.

between control and treated cultures in particle elaboration/celland the apparent absence of cell disintegration on electron microscopic examination. The release of particles having ATPase activity by both control and treated cultures appeared to be parallel. It may be seen in Chart 2, however, that when a decline inthe number of total cells occurred at 21 or 24 hr, indicating disintegration of cells, the ATPase activity rose, deviating from thenormal relation of virus output to cell number. This rise inATPase activity at the end of the experiment might be explainedas being due to cell debris or, possibly, to liberation of intracellu-

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Kosemarie Zischka, A. J. Langlois, P. R. Rao, R. A. Bonar, and J. W. Beard

experiment, also, there was marked inhibition of both cell andvirus RNA synthesis, Table 2.

It is notable, particularly, in Chart 4, that, as in Chart 2,ATPase-positive material increased despite cell damage and decline in virus RXA synthesis. In C'hart 4, there was, indeed, a

substantial and progressive increase in output of particulatematerial with ATPase activity above that in the control cultures.Liberation of such particles in the AD-treated cultures of Chart 5paralleled that of the controls.

Discussion

The myeloblast-BAI strain A virus culture system exhibitssome features different from those of most other RXA virus-cellrelationships. Each myeloblast from the leukemic bird is presumably infected with virus, and the association results in anenduring process of exponential cell growth and virus synthesisand liberation at constant rat es/cell/hr. Cells growing in suspension are constantly available for repeated sampling of thesame preparation, and virus production can be measured in termsof physical particle number by enzyme activity or direct electronmicroscopic count. The myeloblasts investigated were derivativesof cells infected for periods of weeks, and thus the processes requisite for cell growth and virus formation were fully established.Uninfected myeloblasts do not survive in vitro under the conditions employed (8).

Effects of AU on the myeloblasts were exerted on cell growthand on cell RXA synthesis in proportion to the concentration ofAI) in relation to cell concentration. AD in 0.001 MR/m' concentration did not affect cell growth for 15 days, but higher concentrations caused progressive damage resulting in the onset of celldeath after decreasing intervals of exposure (Table 1; Charts 1,2, 4). Sensitivity to the antibiotic varied with cells from differentdonor birds and differed greatly with individual elements of asingle population as indicated by progressive cell death and byvariations in ultrastructural alterations in the later stages of exposure to AD (22). Events occurring with 0.5 /ig AD/ml wererather uniform in the intervals of 5-6 hr treatment (Charts 2, 4)during which time discernible ultrastructural alterations werelimited to changes in the nucleolus.

Cell RXA synthesis was likewise affected by the same factors,but inhibition did not closely parallel changes in cell behavior expressed as growth or survival. Exposure of 3-5 X IO7myelo-

blasts/ml to AD in concentrations of 0.01, 0.05 (45), and 0.5 /¿g/ml resulted in inhibition of uridine-3H incorporation into cell

RXA of about 30, 58, and 86%, respectively. At 0.01 MS/ml,however, growth continued for 6 days (Chart 1); at 0.05 jug/mlfor 48 hr; and at 0.5 us/ml with about 10*cells for 6 hr (Chart 4)or, occasionally, to 21 hr (Chart 2). Thus, damage to the cells evidenced both by inhibition of RXA synthesis and by ultrastructural alterations did not suffice to suppress growth of all cells inthe periods indicated. Nevertheless, exposure to 0.5 MK/mlleadsafterward to progressive cell death with inhibition of cell RXAsynthesis at levels of 60 to 70' „•which were not greatly differentfrom the 58% value found with 0.05 Mfï/mlat the lower cell concentration (45).

A considerable disparity was observed on comparison of cellbehavior with synthesis of virus RXA and liberation of the agent.

As reported (45), virus RXA synthesis was not inhibited by 0.05¿igAD/ml despite a decrease of about 60% in cell RNA synthesis.Instead, the data suggested an increase in virus RNA synthesis.However, treatment with 0.5 jug/ml resulted in comparable inhibitions of virus and cell RXA syntheses. It is notable that themost significant measurements (Table 2) were made in theperiod (Chart 5) before onset of cell death as detected by stainingwith trypan blue and ultrastructural alterations. This indicatedthat inhibition of virus RNA synthesis occurred in cells whichwere viable by the criteria applied.

In contrast to the inhibition of cell and virus RNA syntheseswas the lack of evidence of concurrent suppression of virus particle formation as estimated by ATPase activity. On the contrary, it was consistently seen that the rate of virus particleliberation either remained constant or increased (Charts 2, 4, 5)even after onset of cell death and disintegration.

The findings were explicit in demonstrating inhibition of virusas well as cell RNA syntheses by AD in sufficient concentration.It was further evident, however, that the processes of virus RNAsynthesis were less sensitive to AD than those of cell RNA synthesis, being affected only at higher drug concentrations. An apparently somewhat similar situation has been reported in the caseof reovirus in L cells (34) in which cell RNA synthesis could beinhibited as much as 90% without diminishing virus formation.Differential sensitivities of synthesis of various cell RNA's have

likewise been reported (25, 29).In some respects, AD inhibition of BAI A virus RNA synthe

sis by myeloblasts was analogous to inhibition of Rous viruselaboration by chick embryo fibroblasts (2, 36, 37, 40, 41). Mostof these latter studies (36, 37, 40) were made under conditionsconducive to severe cell damage but, more recently, similar results were obtained (2) with cells not showing morphologic alterations. The experiments with the myeloblast-BAI A virus systemshowed that inhibition could occur in cultures of viable and growing cells which, nevertheless, all showed nucleolar damage (22).Myeloblasts appeared to be less sensitive to AD than chick embryo fibroblasts. This may account for the reported inhibition ofBAI A virus RXA synthesis by fibroblasts (1) treated with Al) in0.005 MS/1*1'concentration in comparison with no inhibition ofBAI A virus RXA synthesis by myeloblasts exposed to 0.05 jugAD/ml.

The basis for continued increase in the number of ATPase-positive particles despite cell damage and partial suppression ofvirus RXA synthesis is not clear from the data thus far obtained.The particles representing increase might have been derived byfragmentation of the plasma membranes of disintegrating myeloblasts which, like the virus, contain ATPase (39). Nevertheless,several aspects of the data: (a) close correspondence between control and treated cultures in the rates of formation of the ATPase-positive particles (Chart 2); (6) the agreement between ATPasedetermination and direct particle count (Table 1); and (c) thesimilarity of sedimentation properties of enzyme activity in thefluids of the control and treated cultures suggested that the particles liberated by the treated cells were similar to the virus. Formation of the agent involves budding of the cell membrane (26)stimulated by thus far unknown mechanisms. Even with inhibition of viral RNA synthesis, continued elaboration of virus par-

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Adinomycin D and RNA Synthesis

tides might be possible, due to the presence of small precursorpolynucleotides accumulated in the infected myeloblasts (35).Further work is necessary to determine whether such particlescontain RXA and exhibit other properties of the virus.

AD inhibition of RSV formation by chick embryo cells hasbeen interpreted to indicate that DNA plays a role as templatefor virus RNA synthesis (2, 36, 37, 40, 41). This view was supported by the observation of homology between RSV RNA andRons cell DNA (38) as well as depression of virus formation byinhibitors of DNA synthesis administered at the time of infection (2, 3, 36, 40, 41). The latter studies were not feasible with theHAI A-myeloblast system because of the lack of a suitable unin-fected cell preparation.

Despite the similarity of some features of AD effects on RSVand HAI A virus RNA syntheses, it seems clear, particularlyfrom recent work with myxoviruses (4, 5,13, 21), that the resultsof studies using AD inhibition as an indicator of DNA-directedviral RNA synthesis must be interpreted with considerable caution. This is emphasized by a variety of observations: (a) there isa very large difference in sensitivity to AD of fowl plague virusformation measured in 2 cell types (100 times more sensitive inchick embryo cells than in allant oie cells) (5); (6) influenza vims-induced RNA synthesis may be less strongly inhibited than formation of infectious particles or hemagglutinin (13, 21); and (c)influenza virus, the elaboration of which is relatively sensitive toAD treatment (6, 21), apparently induces formation of a newribonuclease-sensitive RNA synthetase (18) in calf kidney cells.These findings all suggest that while DNA function is involvedin some way in the formation of these agents, as it is ultimatelyin all cellular activity, its role may well be other than as templatefor viral RNA synthesis. The observation that an RNA-synthe-sizing system using reovirus RNA as template is AD sensitive(19) adds a further dimension to the problem of interpretation,as does the report of other effects of AD (23) apparently notdirectly related to RNA synthesis. When viral RNA synthesisis unaffected by AD, it appears probable that the synthesis doesnot require a DNA template, but the converse cannot be assumed.

References

1. Allen, 1). W., and Zamecnik, P. C. Actinomycin D Sensitivityof UNA Virus (AMV) Production in Chick Embryo Fibroblasta.Federation Proc., $4: 287, 19(15.

2. Bader, J. P. The Role of Deoxyribonucleic Acid in the Synthesis of Rous Sarcoma Virus. Virology, ig: 462-68, 1904.

3. - —.The Requirement for DNA Synthesis in the Growth ofUous Sarcoma and Rons-associated Viruses. Ibid., 26: 253-61,19(15.

4. Barry, R. 1). The Effects of Actinomycin 1) and UltravioletIrradiation on the Production of Fowl Plague Virus. Ibid., 24:563-09, 1964.

5. — —. Effects of Inhibitors of Nucleic Acid Synthesis on theProduction of Myxoviruses. In: G. K. W. Wolstenholme andJ. Knight (eds.), Ciba Foundation Symposium, Cellular Biology of Myxovirus Infections, pp. 51-71. Boston: Little,Brown & Company, 1964.

6. Barry, R. D., Ivés,1). R., and Cruickshank, J. G. Participation of Deoxyribonucleic Acid in the Multiplication of Influenza Virus. Nature, 194: 1139-40, 1962.

7. Beard, J. W. Avian Virus Growths and Their Etiologic Agents.Advan. Cancer Res., 7: 1-127, 1963.

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1966;26:1839-1846. Cancer Res   Rosemarie Zischka, A. J. Langlois, P. R. Rao, et al.  

in VitroVirus RNA Synthesis Effects of Actinomycin D on Avian Myeloblast and BAI Strain A

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