Previous work has determined that the carcinogen4-nitroquinoline N-oxide produces distinctive morphologic changes in Chang liver cells (19). These changesinclude : (a) nucleolar exhaustion manifested by a progressive decrease in nucleolar size and the occasionalfusion of nucleoli, (b) redistribution of the componentsof the nucleolus to produce two types of nucleolar “caps,―and (c) production of intranuclear inclusions. Sincethese alterations in nucleolar morphology may representa morphologic marker for a specific biochemical reaction,other compounds having properties similar to 4-nitroquinoline N-oxide were studied and compared. Theeffects of three of these compounds, actinomycin D,nitrogen mustard, and 2 ,4-dinitrophenol, are presentedin this report.

i\IATERIALS AND METHODS

Chang liver cells in tissue culture were used for allexperiments. They were grown in Eagle's basic medium'with either 10 per cent horse or calf serum (the type ofserum had no influence on the results of the experiments).The cells were grown in tissue culture chambers2 and on1-inch round coverslips in ointment jars. The chambersreceived inoculations of 0.7 cc. and the jars of 3 cc. ofculture medium containing 50,000 Chang liver cells percc.

Growth medium was removed from the jars and chambers and replaced with Hanks balanced salt solutionwhich contained either 4-nitroquinoline N-oxide,3 actino

S This research was supported by the Tobacco Industry Re

search Committee, 4 8778, and the Damon Runyon MemorialFund for Cancer Research@ 8770.

I Obtained from Microbiological Associates, 4813 Bethesda

Avenue, Bethesda 14, Maryland.2 Obtained from Electro-Mechanical Development Co., 2337

Bissonnet Avenue, Houston, Texas.3 Provided through the generosity of Dr. Hideya Endo, Cancer

Institute, Japanese Foundation for Cancer Research, Tokyo.Received for publication March 9, 1964.

mycin D, nitrogen mustard, or 2 ,4-dinitrophenol. Thesechemicals were utilized in concentrations of 10@ M,1O@ M, 10@ M, and 10_6 M. The various dilutions oftest chemicals were allowed to remain on the cells forperiods of time varying from 10 to 20 minutes. Followingthis period of exposure the test solutions were removedand replaced by growth medium. Phase-contrast microscopic images of the cells in tissue culture chambers werethen recorded by means of cinematography. At intervals of 0.5, 1, 2, 4, and 6 hours the coverslip preparationswere removed from ointment jars and fixed for electronmicroscopy and cytologic staining. Dalton's solution and2 per cent potassium permanganate in distilled water (14)were used as fixatives for electron microscopy. Following fixation the cells were dehydrated in graded alcoholsand embedded in methacrylate. Coverslip preparationsfor light microscopic studies were fixed in Carnoy's solution and stained with hematoxylin and eosin, azure “B,―or acridine-orange (1).

Additional procedures were carried out with nitrogenmustard, because the biological effects of nitrogen mustard are apparently due to the development of hydrolysisproducts produced when the chemical is dissolved in anaqueous medium. Golumbic et at. (10) have shown thatthese active hydrolysis products reach a peak concentration in 30—60minutes after nitrogen mustard is dissolvedin an aqueous medium. A 10' M stock solution of nitrogen mustard was prepared in Hanks balanced salt solution.This solution was allowed to remain at room temperaturefor periods of time varying from 5 to 60 minutes, was thendiluted to 10-i M or 10@ M with Hanks balanced salt solution, and the diluted solution was introduced immediatelyinto the cell cultures. Subsequent steps were identicalto those described previously.

RESULTS

In living cells the principal morphologic effects producedby 4-nitroquinoline N-oxide and actinomycin D were

1269

Nucleolar “Caps―Produced by Actinomycin D*

1{. C. REYNOLDS, P. O'B. MONTGOMERY, AND BEN HUGHES

(Department of Pathology, University of Texas Southwestern Medical School and Woodlawn Ho8pital, Dallas, Texas)

SUMMARY

Actinomycin D produced specific morphologic alterations in the nucleoli and nucleiof Chang liver cells in tissue culture. These changes consisted of: (a) a progressive decrease in the size of the nucleoli, (b) redistribution of the components of the nucleolusto produce two types of nucleolar “caps,―and (c) production of intranuclear inclusions.These nuclear changes were identical to those produced by the carcinogen 4-nitroquinoline N-oxide. Nuclear and nucleolar changes of this type were not produced bynitrogen mustard or by 2 ,4-dinitrophenol. These findings suggest that 4-nitroquinoline N-oxide, like actinomycin D, may have a specific effect on the synthesis ofDNA-dependent RNA.

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1270 Cancer Research Vol. 24, August 1964

found in the nuclei and nucleoli. The changes producedby these two agents were virtually identical.

Nucleolar size.—4-Nitroquinoline N-oxide and actinomycin D in concentrations of 10@ M rapidly produced amarked decrease in the size of the nucleoli which wasbest demonstrated with the azure “B―stain. This decrease in the size of the nucleoli was complete within 2hours after application of the chemicals (Figs. 1—3).In addition to losing a considerable proportion of theirmass, the nucleoli also lost their usual irregular contourand assumed a uniform spherical shape. The number ofnucleoli was slightly reduced. With 4-nitroquinolineN-oxide the reduction in the number of nucleoli was duenot only to the loss of mass but also to the occasionalfusion of nucleoli. This phenomenon has been demonstrated by means of time-lapse motion picture studies(19). No fusion of the nucleoli was observed with actinomycin D. Azure “B―and hematoxylin and cosin stainsdemonstrated that the size and contour of Chang livercell nucleoli were unchanged following exposure to nitrogen mustard or 2 ,4-dinitrophenol.

Nucleolar “caps.―—4-NitroquinolineN-oxide and actinomycin D produced striking morphologic changes inthe nucleoli of Chang liver cells. These changes involveda redistribution of nucleolar components to produce thestructures designated as nucleolar “caps.―

Figures 9 and 10 are pictures of normal Chang livercells with phase and electron microscopy. With thephase microscope the nucleolus was frequently irregularand completely solid. On other occasions, as demonstrated in Figure 10, numerous small vacuoles were seen.A previous report has demonstrated that these clearareas are not simple vacuoles but consist of a definitemorphologic component of the nucleolus distinct fromthe surrounding nucleoplasm (19).

Electron photomicrographs of the normal Chang livernucleolus demonstrate the various morphologic patternsnoted by Davis (3) in the nucleoli of normal rat liver cells.The nucleoli consist of a compact mass of particles whichvary considerably in density and measure about 100 A indiameter (Figs. 8, 9). The nucleoli may be solid, vacuolated, or show a skeinlike network in which the denserparticles form strands or cords surrounding clear vacuoles.Occasionally irregular margination of the darkest particbs in the nucleolus may be seen.

The alterations in nucleolar structure produced by 4-nitroquinoline N-oxide and actinomycin D could be seenas early as 30 minutes after removal of the test compounds.The first observed change consisted of a condensation ofthe darker particles to produce electron-dense plaquesscattered irregularly throughout the nucleolus (Figs. 13,17).

Within 1—2hours the electron-dense plaques appearedto migrate toward the margin of the nucleoli, wherethey form an irregular black rim (Figs. 14, 18). Two to4 hours after treatment the marginated particles appearto coalesce into larger masses forming the dark nucleolar“caps―(Figs. 15, 19). These dark nucleolar “caps―have been observed with the phase microscope as wellas with the electron microscope. They have a concavebase and appeared to be pressed or applied to the surface

of the main body of the nucleolus (Figs. 11, 12). Theywere generally multiple and frequently gave the nucleolus a “mulberry―appearance.

A second type of nucleolar “cap―may also be seen inChang liver cells treated with 4-nitroquinoline N-oxideand actinomycin D. This structure has a “nipple-like―appearance. It is composed of particles lighter thanthose forming the dark nucleolar “cap.―This structurehas been designated the light nucleolar “cap―(Figs. 11,16, 20). They were not seen as frequently as the darknucleolar “cap―and were found occasionally in untreatedcells. They were closely attached to the main body ofthe nucleolus and had an indistinct, convex, rather thanconcave, base which protrudes slightly into the nucleolus.The margin between the light nucleolar “cap―and thenucleolus was not as distinct as the margin between thedark “caps―and the nucleolus. When studied with theelectron microscope the light nucleolar “caps―are generally lighter than the main body of the nucleolus, butoccasionally may be composed of darker particles (Fig. 20).These light “caps―were generally single and give thenucleolus an oval shape.

Phase time lapse motion picture studies indicate thatthe light nucleolar “caps―originate from the light “vacuolated―areas of the normal nucleolus. Sequential photographs from a time lapse motion picture demonstratingthe formation of the light and dark nucleolar “caps―may be found in a previous communication (19).

Nucleolar “caps―have not been observed in cellstreated with nitrogen mustard or with 2 ,4-dinitrophenol.Nitrogen mustard produced an occasional small “taillike―nucleolar structure of light particles of about thesame size as the nucleolar “caps.― These structureswere infrequent and did not have the same distributionor morphology as the nucleolar “caps―produced byactinomycin D and 4-nitroquinoline N-oxide. It isprobable that they represent a different phenomenon,since the change is not associated with a decrease innucleolar size. No changes of this type have been foundwith 2 ,4-dinitrophenol.

Nuclear changes.—Figures 4 and 5 demonstrate theinclusions or vacuoles produced by 4-nitroquinoline N-oxide and actinomycin D in the nucleoplasm of cells intissue culture. These structures were first described byEndo in studies with 4-nitroquinoline N-oxide (5, 6).Similar morphologic changes have been observed withactinomycin D in this laboratory. The morphology ofthese inclusions varies with the types of fixation andmicroscopy utilized for study. They were not visiblein the living cell when studied with the phase microscope,nor were they visible with the electron microscope incells fixed with Dalton's solution. They were visiblewith the electron microscope in cells which have beenfixed with potassium permanganate and appeared asdistinct spherical areas of decreased density (Figs. 6, 7).

Following fixation in alcoholic fixatives and stainingwith hematoxylin and eosin, these structures were visibleas distinct intranuclear inclusions. With the acridineorange stain and examination with fluorescent microscopy they appeared as vacuoles containing a minutewisp of pink, fluorescing material. Endo's (6) histo

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REYNOLDS et al.—Nucleolar “Caps―Produced by Actinomycin D 1271

chemical studies and studies with acridine-orange fluorescence (19) indicate that these inclusions or vacuolescontain RNA ; they are not produced by nitrogen mustardor 2 ,4-dinitrophenol.

DISCUSSION

These studies were undertaken to elucidate the biochemical lesion responsible for the morphologic changesproduced by 4-nitroquinoline N-oxide in Chang livercells. The chemical agents were chosen because ofmorphologic or biochemical effects shared in commonwith 4-nitroquinoline N-oxide.

The known morphologic and biochemical effects of 4-nitroquinoline N-oxide have been reviewed by Nakahara(15) and Ono (17). 4-Nitroquinoline N-oxide is a relatively simple chemical compound in which the specificmoieties responsible for its carcinogenic activity havebeen elucidated (16).

The known biochemical effects of 4-nitroquinolineN-oxide include : (a) a decrease in intracellular content ofDPN (17, 21), (b) a strong attraction for sulfhydrylgroups (4), (c) an inhibitive effect on glycolysis (8), and(d) a depressive effect on the ATP content of the cell (8).Secondary effects include inhibition of nucleic acid andprotein synthesis (8).

The cytologic effects of 4-nitroquinoline N-oxide inelude : (a) the production of RNA-containing nuclearinclusions (5, 6, 19), (b) nucleolar exhaustion with anassociated decrease in nucleolar size, and (c) reorganization of the nucleolar substance to form nucleolar “caps―(19).

Actinomycin D was chosen for these studies becauseof previous reports describing nucleolar alterations produced in tissue culture by this substance. It has beenshown that actinomycin D, like 4-nitroquinoline N-oxide,produces a marked reduction in nucleolar size (20).Bierling (2) and Journey and Goldstein (11) have notedphase and electron microscopic changes suggesting thatactinomycin D is capable of producing nucleolar changessimilar to the “caps―we have observed with 4-nitroquinoline N-oxide. The present experiment demonstrates thatnucleolar changes produced by actinomycin D are identicalto those produced by 4-nitroquinoline N-oxide in Changliver cells.

The major biochemical effect of actinomycin D is aninhibition of RNA synthesis brought about by a directcombination of the drug with DNA. Apparently, actinomycin D blocks the templates responsible for assembling DNA-dependent RNA (18). In addition, Levy (12)has shown that actinomycin D blocks the transfer ofnucleolar RNA from the nucleolus to the cytoplasm.This latter observation could explain the RNA-containing nuclear inclusions produced by actinomycin D and4-nitroquinoline N-oxide in Chang liver cells.

The effect of actinomycin D on RNA metabolism maybe inhibited by incorporating DNA into the culturemedium (9). Presumably, the actinomycin D combineswith the extracellular DNA, thus reducing the actinomycin D available for combination with nuclear DNA.

Nitrogen mustard was chosen for these studies becauseof biochemical effects similar to those of 4-nitroquinoline

N-oxide. It has been demonstrated that nitrogen mustard, like 4-nitroquinoline N-oxide, has a depressive effecton cellular DPN concentrations and glycolysis (7). Theaddition of nicotinamide to cultures treated with 4-nitroquinoline N-oxide prevents the decrease in DPN concentration and inhibition of glycolysis (17). Both nitrogenmustard and 4-nitroquinoline N-oxide decrease the ATPcontent of tissue culture cells. This effect is presumablysecondary to decreased DPN levels in the treated cell.The observation that nitrogen mustard did not producethe same type of nucleolar lesions as those produced by4-nitroquinoline N-oxide suggests that the decreasedcellular DPN concentration is not responsible for thenucleolar changes described in this paper.

2 ,4-Dinitrophenol was used in these experiments todetermine whether a decrease in ATP levels, without anassociated decrease in DPN, could be responsible for thenucleolar “caps―and nuclear inclusions. 2 ,4-Dinitrophenol exerts its depressive effect on ATP levels by meansof a specific uncoupling effect on oxidative phosphorylation (13). Since 2 ,4-dinitrophenol did not produce thenuclear or nucleolar changes, it would appear that decreased cellular ATP is not responsible for the morphologic changes produced by 4-nitroquinoline N-oxide andActinomycin D.

In a previous publication the following working hypothesis was proposed as a possible explanation for the effectsof 4-nitroquinoline N-oxide on the nuclei and nucleoliof Chang liver cells: (a) As a result of depressed intracellular DPN and ATP levels, protein and nucleic acidsynthesis in the cell is inhibited. (b) The nucleolus continues to discharge preformed RNA to the cytoplasm in

an attempt to restore adequate protein synthesis. Thecontinued discharge of preformed nucleolar RNA without associated synthesis of RNA results in nucleolarexhaustion, decreased nucleolar size, and redistributionof nucleolar components to form nucleolar “caps.―TheRNA-containing nuclear inclusions were thought torepresent RNA in transit from the nucleolus to thecytoplasm. Complete exhaustion of the available nucicolar RNA is accompanied by cellular death. The presentexperiments with nitrogen mustard and 2 ,4-dinitrophenolindicate that depressed cellular DPN and ATP levels arenot responsible for the nuclear changes observed with4-nitroquinoline N-oxide. The observation that actinomycin D produces nucleolar exhaustion, nucleolar “caps,―and nuclear inclusions suggests that 4-nitroquinolineN-oxide, like actinomycin D, may specifically inhibitsynthesis of RNA. The studies of Levy (12) suggest thatthe nuclear inclusions do not represent RNA in transitto the cytoplasm but, rather, RNA trapped in the nucleoplasm by an inhibiting effect of these drugs on the transport of RNA from the nucleolus to the cytoplasm. Iffuture studies prove this to be true, then the observedchanges in the nucleoli and nuclei of Chang liver cellsmay, in fact, represent reproducible morphologic markersfor specific biochemical reactions—i.e., the inhibition ofsynthesis of DNA-dependent RNA and the transfer ofavailable nucleolar RNA from the nucleolus to the cytoplasm.

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1272 Cancer Research Vol. 24, August 1964

mations of Methyl-Bis(@-Ch1oroethy1) Amine in Water. JOrg. Chem., 11:518—35,1946.

11. JOURNEY, L. J., ANDGOLDSTEIN,MILTON N. Electron Microscope Studies on HeLa Cell Lines Sensitive and Resistant toActinomycin D. Cancer Res. , 21 :929-32, 1961.

12. LEVY, H. B. Effect of Actinomycin D on HeLa Cell NuclearRNA Metabolism. Proc. Soc. Exp. Biol. Med., 113:886—89,1963.

13. LooMis, W. F., ANDLIPMANN, F. Reversible Inhibition of theCoupling between Phosphorylation and Oxidation. J. Biol.Chem., 173:807—8,1948.

14. MOLLENHAUER, H. H., AND ZEBRUN, W. Permanganate Fixation on the Golgi Complex and Other Cytoplasmic Structuresof Mammalian Testes. J. Biophys. Biochem. Cytol., 8:761—75,1960.

15. NAKAHARA,W. Critique of Carcinogenic Mechanism. Prog.Exp. Tumor Res.,2:158—202,1961.

16. NAKAHARA,W.; FUKUOKA,F.; AND SAXAI, S. The Relationbetween Carcinogenicity and Chemical Structure of CertainQuinoline Derivatives. Gann, 49:33—41,1958.

17. ON0, T.; TOMARU, T.; AND FUKUOKA, F. Effect of 4-Nitroquinoline N-oxide Derivatives on the DPN Dependent Enzyme Systems. Gann, 50:189-200, 1959.

18. REICH, E.; FRANKLIN, R. M.; SHATKIN, A. J.; AND TATUM,E. L. Effect of Actinomycin D on Cellular Nucleic AcidSynthesis and Virus Production. Science, 134:556—57,1961.

19. REYNOLDS,R. C.; MONTGOMERY,P. O'B.; ANDKARNEY, D.H. Nucleolar “Caps―—AMorphologic Entity Produced bythe Carcinogen 4-Nitroquinoline N-oxide. Cancer lIes.,23:535—38,1963.

20. ROUNDS, D. E.; NAKANISHI, Y. H.; AND POMERAT, C. M.Possible Mechanisms To Explain the Action of Actinomycinon Non-malignant and Malignant Cells. Antibiotics & Chemotherapy, 10:597-603, 1960.

21. TOMARU,T.; OHASHI,M.; ANDFUKUOKA,F. Effect of 4-Nitroquinoline N-oxide on the DPN Metabolism of Tumor Tissue.Gann, 52:89—95, 1961.

ACKNOWLEDGMENTS

Grateful appreciation is extended to Dale McClendon, BettyStapp, and Louise Stinson for their assistance in tissue culture,photography, and electron microscopy.

REFERENCES

1. BERTALANFFY,L.; MASIN, M. ; ANDMASIN, F. A New andRapid Method for Diagnosis of Vaginal and Cervical Cancerby Fluorescence Microscopy. Cancer, 2:873—87,1958.

2. BIERLING, R. Die Wirkung von Actinomycinen auf Menschliche Gewebe in vitro. Z. Krebsforsch., 63:519—22,1960.

3. DAVIS, J. M. G. The Ultra@tructure of the Mammalian Nucleolus. In: J. S. MITCHELL(ed.), Cell Nucleolus, Chapter I.New York: Academic Press, 1960.

4. ENDO,H. On the Relation between Carcinogenic Potency of4-Nitroquinoline N-Oxides and the Reactivity of TheirNitro-groups with S-H Compounds. Gann, 49:151-56, 1958.

5. ENDO, H. ; Aoxi, M. ; ANDAOYAMA,Y. Formation of NuclearInclusion Bodies in Tissue Culture Cells by 4-NitroquinolineN-oxide. Gann, 50:209—17,1959.

6. ENDO, H.; TAKAYAMA,S.; KASUGA,T.; AND OHASHI,M.Histochemical Studies on Nuclear Inclusion in Tissue CultureCells Induced by 4-Nitroquinoline N-oxide. Gann, 52:173-77,1961.

7. FRASER, I. M. Effect of Mechiorethamine on Tumor Glycoly515 and Diphosphopyridine Nucleotide Content. Fed. Proc.,19:395, 1960.

8. FUKUOKA, F.; ON0, T.; OHASHI, M.; AND NISHIMUJIA, S.Effect of 4-Nitroquinoline N-oxide on the Metabolism ofCancer Cellsin vitro.Gann, 50:(1)23—25,1959.

9. GOLDBERG,I. H., ANDR@t.rnNowiTz,M. Actinomycin D Inhibition of Deoxyribonucleic Acid-Dependent Synthesis ofRibonucleic Acid. Science, 136:315—16,1962.

10. GOLUMBIC,C. ; FRUTON,J. S. ; ANDBERGMANN,MAX. ChemicalReactions of the Nitrogen Mustard Gases. I. The Transfor

FIG. 1.—Untreated Chang liver cells stained with azure “B.―Note size and irregularity in the shape of the nucleoli (N). Mag.X3t50.

Fm. 2.—Chang liver cells 2 hours after being treated with10—bM 4-nitroquinoline N-oxide for 10-15 minutes. Note thedecrease in size of the nucleoli (N) and their uniform sphericalshape. Azure “B―stain. Mag. X350.

Fia. 3.—Chang liver cells 2 hours after being treated with10—sM actinomycin D for 10—15minutes. Note the decrease insize of nucleoli (N) and uniform spherical shape. Azure “B―stain. Mag. X350.

FIG. 4.—Intranuclear inclusions (NI) produced by 10' M4-nitroquinoline N-oxide in Chang liver cells. Carnoy's fixative.Hematoxylin and eosin (H. & E.) Mag. X550.

Fia. &.—Intranuclear inclusions (NI) produced by 10@ Mactinomycin D in Chang liver cells. Carnoy's fixative. H. &E., X500.

Fio. 6.—Electron microscopic appearance of intranuclear inclusions (NV) produced by 4-nitroquinoline N-oxide followingfixation in potassium permanganate. These structures are believed to correspond to the nuclear inclusions seen in Figs. 4 and5. The spherical areas of decreased density are not visible withosmium tetroxide fixatives, such as Palade's solution. Comparewith normal Chang liver cell (Fig. 8). Mag. X5,800.

FIG. 7.—Electron photomicrograph of intranuclear inclusions(NV) produced by 10@Mactinomycin D. Potassium permanganate fixation. Mag. X7,500.

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Fio. 8.—Electron photomicrograph of normal Chang liver cell.Potassium pernianganate fixation. Note lack of the large spherical areas of decreased density which are present in cells treatedwith 4-nitroquinoline N-oxide and actinomycin D (Figs. 6, 7).Mag. Xll,600.

Fxo. 9.—Normal Chang liver cell. Palade's fixative. Notegranular pattern and vacuolization of nucleolus (N). Mag.X8,700.

F:o. 10.—Phase photomicrograph of m)rnlal Clang livernucleolus (N). Mag. X3,500.

Fir. 11.—Phase photon@icrograph of light nucleolar “caps―(L'VC) and dark nucleolar “caps' †(DNC) produced by l0@ M4-nitroquinoline \-oxide in Chang liver cells. Mag. X3,500.

Fio. 12.—Phase photoniicrograph of dark nucleolar “caps―(I).\@(') produced by l0@ M actinomycin 1) in Chang liver cells.Mag. X3,500.

1274

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FIGS. 13—16.—Electron photonhicr()graphs of Chaiig 1iver cellstreated with 4-nitroquinoline A-oxide. PaIa(le's fixative.

Fin. 13—Nucleolus 1 hour after exposure to 10@ M 4-nitro(itlinoline A-oxide for 10—15 iniiiutes. Note condensation ofelectron-dense particles to form nucleolar plaques (NP) scatteredthroughout the nucleolus. Mag. X5,000.

FIG. 14.—Appearance of Chang liver cells 1?@—3hours afterexposure to 10@ M4-nitroquinoline .V-oxide.—Note that nucleolarplaques (.VP) appear to have migrated to periphery of nucleolusto forni dark nucleolar “caps― (DNC). Mag. X20,000.

Fu,. 15.—Four hours after exposure to 10@ M 4-nitroquinoline.\-oxide.—Note further condensation of dark niicleolar ‘‘caps―(I)X(') to foriii l)ulging masses wit Ii concave bases, sharply (ICniarcated from nucleolus proper. Mag. XS,209.

@ 16.—Light (L.V(') and dark nucleolar “caps'â€(J).V(') 4hours after exposure to l0' M 4-nitroquinoline .\-oxide. Thelight nucleolar ‘‘(-aps''are usually single tuid have convexand the dark nucleolar ‘‘caps'â€are niult iple atid usually haveconcave bases. Mag. Xll,600.

Fu;s. 17—20.—Electron photoinicrographs of Chang liver cellst reated wit h act inotnyc iii I ) . Palade ‘sfixat 1VC.

Fin. 17.—Nucleolus 3@ minutes after exposure to 10@ M act moinycin I) for 1Q—15n@inutes. Note fornlat ion of tiucleolar l)lallles

(XV). Mag. X6,600.Fio. 18.—Chang liver cell 1 hour after exposure to 10@ M

act i0011ivcin I ). Note that nucleolar plaques (.VP) appear tohave migrated to 1)eripherY of nucleus to form dark nucleolar“caps―(D.V('). Mag. X13,200.

Fio. 19—Two to 4 hours after exposure to 10@ M actinoinycinI). Note further condensati(@n of dark nucleolar ‘‘caps''(I).V(')to pr@(luce a ‘‘mulberry-like' â€nucleolus. )@Iag. X 11,(@00.

Fin. 20.—Light (L@\'() and dark (D.V€') nucleolar “caps―produced l@' actinoinycin 1). Note t he convex base of the lightnL1cle(@lar ‘‘caps''(L.\('). In this instance the light nticle,lar‘‘cap'â€is darker t littn t he main port ion of tl@e nucleolus. Mag.x 19,200.

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1964;24:1269-1277. Cancer Res   R. C. Reynolds, P. O'B. Montgomery and Ben Hughes  Nucleolar ''Caps'' Produced by Actinomycin D

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