[CANCER
RESEARCH
27, 1232-1242, July 1967]
Formation of Microspherules in Nucleoli of Tumor Cells Treated
with High Doses of Actinomycin D1
TADAO
UNUMA
AND HARRIS BUSCH
Department of Pharmacology, Baylor University College of Medicine, Houston, Texas 770Z5
SUMMARY
Microspherules, composed of condensed and segregated fibrillar
components, were observed in the nucleoli of the Ehrlich ascites
cells and in those of the Novikoff hepatoma cells after treatment
with high doses of actinomycin D, but not in Walker tumor cells.
The number of these microspherules was calculated to range from
100 to 300 per nucleolus. The density of the microspherules de
creased markedly after treatment with pepsin and ribonuclease,
but not after treatment with deoxyribonuclease. The microspherules contain both protein and RNA and are not aggrega
tions of chromatin as evidenced by their lack of DXA. In the
Walker tumor cells, compact nucleolonemas, segregation, and a
decreased amount of granular components and vacuole-like areas
were observed in the nucleoli after treatment with actinomycin
D.
INTRODUCTION
Biochemical and morphologic studies on the effect of actino
mycin D have revealed its selective suppressive effect on bio
synthesis of nucleolar RNA (4, 5, 12, 16, 19, 21-23, 26-28, 41)
which is accompanied by characteristic morphologic changes such
as: interior hyalinization, dense peripheral granules and frag
mentation of the nucleoli in HeLa cells (10); decrease in the size
of nucleoli and the two types of nucleolar caps in Chang liver
cells (24) ; buds and satellites in the cultured baboon kidney cells
(30); loss of granules, centrally located light islet, and new periph
eral substance in the salivary gland cells of Chironomus thummi
(39) ; complete loss or reduction in size of the nucleoli of Smittia
parthenogenetica (7), fragmented nucleoli in starfish oocytes (3);
and segregation of the components of the nucleoli and furrow
between the nucleolus and chromatin (9) in the rat liver and
pancreas (9, 31, 35-38), and also in avian leukemic myeloblast
(6).
Since actinomycin I) inhibits the synthesis of rapidly sedimenting nuclear RXA (17-19) and increases the proportion of
adenine- and uracil-rich RNA in the nucleolus (28), the present
study was designed to investigate the morphologic changes in
cancer cell nucleoli, which contain abundant guanine- and cytosine-rich RNA of high molecular weight and granular ribonucleoprotein (15, 35). At the high doses employed, dense bodies were
observed in nucleoli which appear to be microspherules composed
1Supported by grants from the American Cancer Society, the
USPHS (CA-8182), and the Jane Coffin Childs Fund.
Received July 7, 1966; accepted March 7, 1967.
1232
of fibrillar nucleolar elements. These dense microspherules were
observed in nucleoli of Ehrlich and Novikoff ascites cells but
not in Walker tumor nucleoli. Morphologically similar structures
have been found previously after treatment of cells with actino
mycin D (24), aflatoxin B! (1), and other drugs (32, 33).
Digestion studies with pepsin, ribonuclease (RNase), deoxy
ribonuclease (DNase), and combinations of these enzymes were
employed to determine the composition of the microspherules
produced by high doses of actinomycin D. These studies indicate
that ribonucleoprotein but not DNA is present in the microspherules.
MATERIALS AND METHODS
Effects on the Ehrlich ascites cells, 7 days after transplantation
into Swiss male mice (Euers, Austin, Texas), weighing 20 to 25
gm, were studied 60 minutes after intraperitoneal injection of
actinomycin D in doses of 800, 1600, and 3200 Mg/kg body weight.
The effects of the drug were also examined on Novikoff hepatoma
cells transplanted as an ascites tumor in Holtzman rats. These
cells were incubated with actinomycin D in the ascites fluid.
Specimens were obtained 60 minutes after incubation in vitro of
1.7 X IO7cells/ml with actinomycin D in a concentration of 0.5,
2.5, 5, and 10 /¿g/ml,and 60 minutes after intraperitoneal injec
tion of actinomycin D in vivoin doses of 800 Mg/kg body weight.
Samples of Walker 256 carcinosarcoma of rats were obtained 5,
10, 30, and 60 minutes after intravenous administration of 400
and 800 Mg/kg body weight of actinomycin D.
For electron microscopic studies, the tissues were fixed in
6.25 % glutaraldehyde for the Walker tumor cells, or 3% glutaraldehyde for the Ehrlich ascites cells and the Novikoff hepatoma
cells. The glutaraldehyde and the sample were incubated in 0.1
Mphosphate buffer at pH 7.4 for 2 hours at 4°C;postfixation was
carried out in 2% osmium tetroxide in 0.14 M Veronal acetate
buffer at pH 7.4 for 1 hour. Some of the tissues were fixed in 2%
osmium tetroxide for 2 hours and postfixed in 10% formalin in
0.1 M phosphate buffer at pH 7.4 for 1 hour. Dehydration was
carried out with graded concentrations of ethanol containing
uranyl acetate (34). They were embedded in Epon-Araldite (13)
and stained with 2.5% uranyl acetate in 50% ethanol and 0.2%
lead citrate (42). Thin sections were obtained with a LKB ultrotome and were studied with a Philips EM 200 Electron micro
scope.
For enzymatic digestion studies (6, 11, 40), the tissues were
fixed in 10% formalin in 0.1 M phosphate buffer at pH 7.4 for
15 minutes at 4°C,washed twice with 0.1 Mphosphate buffer and
treated with the following enzymes: (a) pepsin twice crystallized,
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Microspherules in Actinomycin D-ireated Tumor Cell Nucleoli
0.01 mg/ml in 0.1 N HCl, 10 minutes; (6) RNase crystallized, 1
mg/ml, pH 6.5, 2 hours; (c) DNase electrophoretically purified,
free of RNase, 1 mg/ml in 0.003 M magnesium acetate, 1 hour;
(d) pepsin followed by RNase treatment; (è)pepsin followed by
DNase treatment. The enzyme preparations used in the present
studies were obtained from Worthington Biochemical Corpora
tion, Freehold, New Jersey. RNase, dissolved in acetate buffer,
pH 5.0, was heated at 80°Cfor 10 minutes to remove DNase
activity and was adjusted to pH 6.5 with 0.01 N NaOH. Before
treatment with the second enzyme, the specimens were washed 3
times with 0.1 M phosphate buffer. Enzymatic reactions were
terminated by incubation in 5% trichloracetic acid for 15 minutes
at 2°C.The specimens were dehydrated, embedded in EponAraldite, and stained with uranyl acetate and lead citrate.
RESULTS
Effects on Ehrlich Ascites Cells, untreated Ehrlich ascites
cells contained large nucleoli, which were composed mainly of
granular components (Figs. 1, 2). Vacuole-like areas were ob
served in some nucleoli (Figs. 1, 2). Perinucleolar nucleolusassociated chromatin and intranucleolar chromatin were evident
in glutaraldehyde-fixed material (Fig. 1), but in osmium-fixed
material, chromatin was extracted and of low electron density
(Fig. 2).
After treatment with actinomycin D, electron-dense microspherules were observed in most nucleoli at each of the doses of
actinomycin D employed, i.e., 800-3200 ng/kg body weight
(Figs. 3-5). These spherules had an electron density similar to
that of the clumped nucleolus-associated chromatin of samples
fixed in glutaraldehyde (Fig. 3). In samples of the same specimen
fixed in osmium tetroxide, the density of the spherules was much
greater than the chromatin (Fig. 4). The spherules had sizes
ranging from 0.05 y. to 0.2 n and had halos of low electron density
around them (Fig. 3), and in some instances, centrally located
light areas were observed at higher magnifications (Fig. 5). At
high magnifications, the spherules contained fine fibrils (Fig. 5),
which were more electron dense than the normal fibrillar com
ponents of the nucleolus. In some nucleoli, the spherules were
located almost exclusively at the periphery of the nucleolus (Fig.
4), and in others, they were found within the nucleolus (Fig. 3)
and located mainly in the granular areas.
Although these morphologic effects of actinomycin D were
found to the same extent in the nucleoli of the specimens taken
from animals treated with 800, 1600, and also 3200 ¿ig/kgbody
weight of the drug, no remarkable changes were seen in the extranucleolar areas of the nucleus or in the cytoplasm.
Effects on Novikoff Hepatoma Cells. The nucleoli of un
treated Xovikoff hepatoma cells were composed mainly of granu
lar components after fixation in glutaraldehyde (Fig. 6), or in
osmium tetroxide (Fig. 7). Nucleolonemas were evident after
both fixation procedures (Figs. 6, 7), but the nucleolus-associated
chromatin and intranucleolar chromatin were more evident in
samples fixed in glutaraldehyde (Fig. 6).
The effects of actinomycin D on the Novikoff hepatoma cells
in vitro and in vivo were quite similar to those on the Ehrlich
ascites cells in vivo. The microspherules found were similar to
those present in Ehrlich ascites cells (Figs. 8-10). These microspherules were observed in specimens fixed in glutaraldehyde
JULY 1967
(Fig. 8), fixed in osmium tetroxide (Fig. 9), and also fixed in
formalin (Fig. 10). Halos were present around the microspherules
(Figs. 8-10). The microspherules were located both at the periph
ery of the nucleolus and within the nucleolus (Fig. 8-10).
In experiment in vitro, the microspherules appeared only in cells
treated with 10 jug/ml of the drug. No remarkable changes were
observed in the extranucleolar areas of the nucleus or in the
cytoplasm.
Enzymatic
Digestion
Studies
Pepsin. Density of the microspherules, produced by high doses
of actinomycin D, was reduced slightly after treatment with
pepsin (Fig. 11). The granular and fibrillar components of the
nucleolus appeared more sharply after removal of a part of the
protein matrix by the pepsin treatment.
RNase. The density of the microspherules was decreased
remarkably after RNase treatment (Fig. 12) and simultaneously
the granular elements of the nucleolus disappeared. The nucleo
lus-associated chromatin was largely intact. In some instances
the microspherules appeared to be adjacent to the nucleolusassociated chromatin.
Pepsin and RNase. With both enzymes, the density of the
microspherules was decreased markedly (Fig. 13) by comparison
with pepsin treatment alone (Fig. 11), but the end result was not
very different from treatment with RNase alone. All the granular
elements of the nucleolus disappeared along with the ribosomes in
the cytoplasm. The pepsin-resistant matrix of the nucleolus, the
nuclear chromatin, and nucleolus-associated chromatin were
apparently intact after treatment.
DNase. The microspherules showed no significant decrease in
their density after DNase treatment (Fig. 14). The granular ele
ments in the nucleolus were intact. The nucleolus-associated
chromatin and the nuclear chromatin were removed by DNase.
Pepsin and DNase. The density of the microspherules was not
decreased markedly (Fig. 15), compared with the specimen
treated withDXase alone (Fig. 14). The granules in the nucleolus
appeared more sharply after pepsin treatment. No nucleolusassociated chromatin was observed, and the nuclear chromatin
was removed. The nuclear ribonucleoprotein network remained
as a residue.
Effects on Walker Tumor Cells. After fixation with glutar
aldehyde, untreated Walker tumor cells contained large nucleoli
which were composed of nucleolonemas containing mainly gran
ular elements (Fig. 16).
Marked effects of actinomycin D on the nucleoli were seen at
60 minutes after administration of 800 MK/kgi-v. of actinomycin
D: (a) in most nucleoli internucleolonemal spaces were lost and
the nucleolus became compact (Figs. 17,18), and (6) the area con
taining granular elements was much decreased and segregated
sharply from the fibrillar elements (Figs. 17, 18); (c) in many
nucleoli, large vacuole-like areas were present (Fig. 18).
No remarkable changes were observed 5 minutes after admin
istration of 400 and 800 Mg/kg body weight of actinomycin D. At
10 minutes after administration of actinomycin D, the changes
were much less marked and granular areas were decreased to a
smaller extent by comparison with the effect at 60 minutes. A
decrease in granular elements and segregation of the components
were evident 30 minutes after administration of actinomycin D in
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Tadao Unuma and Harris Busch
doses of 400 and 800 MgAg of body weight. No changes in the
extranucleolar area of the nucleus and cytoplasm were observed.
DISCUSSION
Microspherules were noted in the nucleoli of the Ehrlich ascites
cells and Novikoff hepatoma cells treated with high concentra
tions of actinomycin D. At low power, these structures are similar
to those found by Reynolds et al. (24) ; they reported that nucleolar plaques were scattered irregularly throughout the nucleolus
30 minutes after treatment with actinomycin D in the Chang
liver cells. Sixty minutes after this treatment, they observed that
the nucleolar plaques migrated to the periphery of the nucleolus
where they might form dark nucleolar caps. The nucleolar plaques
were also observed after irradiation with a shaped microbeam of
low-intensity heterochromatic ultraviolet light (14). Satellites or
blebs (30) were found at the surface of or extruding from nucleoli
of baboon kidneys after actinomycin treatment. These appear to
be different from the microspherules which were embedded within
the nucleolus and had a relatively uniform size. Similar dense
structures were observed after treatment with aflatoxin BI (1)
in rat liver, proflavin (32), ethionine bromide (33), daunomycin
(33), echinomycin (33), and chromomycin (33) in the cultured
rat embryonic cells. Microspherules were also noted in liver
nucleoli of rats treated with both thioacetamide and actinomycin
D (31).
The microspherules which appeared in the nucleoli after treat
ment with actinomycin D were found in the present studies to be
composed of dense, tightly coiled fibrillar components surrounded
by a slight halo and occasionally a light core was present. The
enzyme studies showed that the microspherules contained both
protein and RNA. On the basis of enzyme studies, Shoefl (30)
concluded that "blebs" and satellites of actinomycin-treated
baboon kidney nucleoli contained RNA and protein also. Forma
tion of the microspherules might occur if high doses of the inhibi
tor attacked some auxiliary function in the foci of RNA synthesis
and as a result produce condensation of the fibrillar components
of the foci. Some spherules were associated with the nucleolusassociated chromatin. The number of the microspherules per
nucleolus averaged 180 and ranged from 100 to 300 per nucleolus
in the 12 Ehrlich ascites cells. An average of 220 microspherules
was found in the 9 Novikoff hepatoma cells studied; the range
was 150-310. An interesting correlation may exist between these
numbers of the microspherules and the value of the 240 cistrons
for synthesis of ribosomal RNA derived from earlier studies (25).
Although decreases of granular components of the nucleoli by
actinomycin D were observed in the Walker tumor in the present
experiment, the rates at which morphologic changes were found
were much slower than those at which biochemical changes were
noted (8). Morphologic effects were found at 30 and 60 minutes
after injection of actinomycin D, but the suppressive effects were
noted biochemically within 10 minutes after injection of the drug.
Substantial evidence is available that the 45 S nucleolar RNA is a
precursor of 28 S RNA of the ribosomes (2, 17, 18, 20, 29), but the
relationship of the effects observed in this study to ribosomal
synthesis is not yet defined.
REFERENCES
1. Bernhard, W., Frayssinet, C., Lafarge, C., and LeBreton, E.
LésionsNucléolaires Précoces Provoquées par l'Aflatoxine
1234
dans les Cellules Hépatiques du Rat. Compt. Rend., ¡61:
1785-1788, 1965.
2. Edström, J.-E., and Gall, J. G. The Base Composition of Ribonucleic Acid in Lampbrush Chromosomes, Nucleoli, Nuclear
Sap and Cytoplasm of Triturus Oocytes. J. Cell Biol., 19:
279-284, 1963.
3. Geuskens, M. ÉtudeAutoradiographique
et Ultrastructurale
de l'Action de l'Actinomycine
D sur les Oocytes D'astérie.
Exptl. Cell Res., 39: 400-412, 1965.
4. Goldberg, I. H., and llabinowitz, M. Actinomycin D Inhibi
tion of Deoxyribonucleic Acid-dependent Synthesis of Ribonucleic Acid. Science, 136: 315-316, 1962.
5. Harbers, E., and Müller,W. On the Inhibition of RNA Synthe
sis by Actinomycin. Biochem. Biophys. Res. Commun., 7:
107-110, 1962.
6. Heine, U., Langlois, A. J., and Beard, J. W. Ultrastructural
Alterations
in Avian Leukemic Myeloblasts
Exposed to
Actinomycin D in Vitro. Cancer Res., 26: 1847-1858, 1966.
7. Jacob, J., and Sirlin, J. L. Electron Microscopic Studies on
Salivary Gland Cells. IV. The Nucleus of Smittia parthenogenetica (Chironomidae)
with Special Reference to the
Nucleolus and the Effects of Actinomycin Thereon. J. Ultrastruct. Res., //: 315-328, 1964.
8. Jacob, S. T., Steele, W. J., and Busch, H. Turnover of 458
RNA in the Regenerating liver and the Walker Tumor. Cancer
Res., 27: 52-60, 1967.
9. Jézéquel,
A. M., and Bernhard, W. Modifications Ultrastruc
turales du PancréasExocrine de Rat Sous l'Effet de l'Actino
mycine D. J. Microscop., 3: 279-296, 1964.
10. Journey, L. J., and Goldstein, M. N. Electron Microscope
Studies on HeLa Cell Lines Sensitive and Resistant to Actino
mycin D. Cancer Res., él:929-932, 1961.
11. Leduc, E., Marinozzi, V., and Bernhard, W. The Use of WaterSoluble Glycol-methacrylate
in Ultrastructural
Cytochemis
try. J. Roy. Microscop. Soc., 81: 119-130, 1963.
12. Merits, I. Actinomycin Inhibition of RNA Synthesis in Rat
Liver. Biochem. Biophys. Res. Commun., 10: 254-259, 1963.
13. Mollenhauer, H. H. Plastic Embedding Mixtures for Use in
Electronmicroscopy.
Stain Technol., 39: 111-114, 1964.
14. Montgomery, P. O'B., Reynolds, R. C., and Cook, J. E. Nu
cleolar "Caps" Induced by Flying Spot Ultraviolet Nuclear
Irradiation. Am. J. Pathol., 49: 555-567, 1966.
15. Muramatsu, M., and Busch, H. Studies on Nucleolar RNA of
the Walker 25C Carcinosarcoma
and the Liver of the Rat.
Cancer Res., ¡4:1028-1034, 1964.
16. Muramatsu, M. Hodnett, J. L., and Busch, H. Studies on the
'Independence' of Nucleolar Ribonucleic Acid Synthesis. Biochim. Biophys. Acta, 91: 592-597, 1964.
17. Muramatsu, M., Hodnett, J. L., Steele, W. J., and Busch, H.
Synthesis of 28S RNA in the Nucleolus. Biochim. Biophys.
Acta, 123: 116-125, 1966.
18. Perry, R. P. The Cellular Sites of Synthesis of Ribosomal and
4S RNA. Proc. Nati. Acad. Sei. U. S., 48: 2179-2186, 1962.
19. Perry, R. P. Selective Effects of Actinomycin D on the Intracellular Distribution
of RNA Synthesis in Tissue Culture
Cells. Exptl. Cell Res., 29: 400-408, 1963.
20. Perry, R. P., Hell, A., and Errera, M. The Role of the Nucleo
lus in Ribonucleic Acid and Protein Synthesis. I. Incorpora
tion of Cytidine into Normal and Nucleolar Inactivated HeLa
Cells. Biochim. Biophys. Acta, 49: 47-57, 1961.
21. Reich, E. Biochemistry of Actinomycins. Cancer Res., #3/
1428-1441, 1963.
22. Reich, E. Actinomycin: Correlation of Structure and Function
of Its Complexes with Purines and DNA. Science, 143: 684689, 1964.
23. Reich, E. Franklin, R. M., Shatkin, A. J., and Tatum, E. L.
CANCER RESEARCH
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1967 American Association for Cancer Research.
VOL. 27
Microspherules in Actinomycin D-treated Tumor Cell Nucleoli
24.
25.
26.
27.
28.
29.
30.
31.
32.
Effect of Actinomycin D on Cellular Nucleic Acid Synthesis
and Virus Production. Science, 184: 556-557, 1961.
Reynolds, R. C., Montgomery, P. O'B., and Hughes, B. Nucleolar "Caps" Produced by Actinomycin D. Cancer Res., 84-'
1269-1277, 1964.
Ritossa, F. M., and Spiegelman, S. Localization of DNA Com
plementary to Ribosomal RNA in the Nucleolus Organizer
Region of Drosophila melanogasler. Proc. Nati. Acad. Sei. U. S.,
öS:737-745, 1965.
Ro, T. S., and Busch, H. In vitro Labeling of RNA in Isolated
Nucleoli of the Walker Tumor and Liver. Cancer Res., 84-'
1630-1633, 1964.
Ro, T. S., and Busch, H. Concentration of "C-Actinomycin D
in Various Tissues Following Intravenous Injection. Biochim.
Biophys. Acta, 108: 317-318, 1965.
Ro, T. S., Shankar Narayan, K., and Busch, H. Effects of
Actinomycin D on Base Composition and Nearest Neighbor
Frequency of Nucleolar RNA of the Walker Tumor and Liver.
Cancer Res., S6: 780-785, 1966.
Scherrer, K., Latham, H., and Darnell, J. E. Demonstration of
an Unstable RNA and of a Precursor to Ribosomal RNA in
HeLa Cells. Proc. Nati. Acad. Sei. U. S., 49: 240-248, 1963.
Schoefl, G. I. The Effect of Actinomycin D on the Fine Struc
ture of the Nucleolus. J. TJltrastruct. Res., 10: 224-243, 1964.
Shankar Narayan, K., Steele, W. J., and Busch, H. Evidence
that the Granular and Fibrillar Components of Nucleoli Con
tain 28 and 6S RNA Respectively. Exptl. Cell Res., 43: 483492, 1966.
Simard, R. Specific Nuclear and Nucleolar Ultrastructural
JULY 1967
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
Lesions Induced by Proflavin and Similarly Acting Antimetabolites in Tissue Culture. Cancer Res., 26: 2316-2328, 1966.
Simard, R., and Bernhard, W. Le Phénomène
de la Ségrégation
Nucléolaire: Spécificité
d'Action de Certains Antimétabolites.
Intern. J. Cancer, /: 463-479, 196G.
Smetana, K., and Busch, H. On théUltrastructure
of the
Walker 256 Carcinosarcoma. Cancer Res., 23: 1600-1003, 1963.
Smetana, K., Shankar Narayan, K., and Busch, H. Quantita
tive Analysis of Ultrastructural
Components of Nucleoli of the
Walker Tumor and Liver. Cancer Res., 26: 786-796, 1966.
Smuckler, E. A., and Benditt, E. P. The Early Effects of Acti
nomycin on Rat Liver. Changes in Ribosomes and Polysomes.
Lab. Invest., 14: 1699-1709, 1965.
Stenram, U. Electron-Microscopic
Study on Liver Cells of
Rats Treated with Actinomycin D. Z. Zellforsch., 65: 211219, 1965.
Stenram, U., and Willen, R. The Effect of Actinomycin D on
Ultrastructure
and Radioautographic
Ribonucleic Acid and
Protein Labeling in Rat Liver after Partial Hepatectomy.
Cancer Res., 86: 765-772, 1966.
Stevens, B. J. The Effect of Actinomycin D on Nucleolar and
Nuclear Fine Structure in the Salivary Gland Cell of Chironomus thummi. J. Ultrastruct. Res., 11: 329-353, 1964.
Swift, H. Cytochemical Studies on Nuclear Fine Structure.
Exptl. Cell Res., 9 (Suppl.): 54-67,1963.
Tamaoki, T., and Mueller, G. C. Synthesis of Nuclear and
Cytoplasmic RNA of HeLa Cells and Effects of Actinomycin
D. Biochem. Biophys. Res. Commun., 9: 451-454, 1962.
Venable, J. H., and Coggeshall, R. A Simplified Lead Citrate
Stain for use in Electronmicroscopy.
J. Cell Biol., So: 407-408,
1965.
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Tadao Unuma and Harris Busch
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FIGS. 1-18. The marker line in the figures denote one micron./, fibrillar components of the nucleolus; g, granular components of the
nucleolus; c, nucleolus-associated chromatin; v, vacuole-like area of the nucleolus; s, microspherules in the nucleolus. Specimens were
embedded in Epon-Araldite and stained with uranyl acetate and lead citrate.
FIG. 1. Nucleolus of an untreated Ehrlich ascites cell. Nucleolus-associated chromatin (c) is apparent. A vacuole-like area (v) is
present. The specimen was fixed in glutaraldehyde and postfixed in osmium tetroxide. X 23,000.
FIG. 2. Same specimen as in Fig. 1, but fixation was carried out in osmium tetroxide and formalin. The chromatin is extracted.
X 26,000.
FIG. 3. A nucleolus of an Ehrlich ascites cell, 60 minutes after administration of 1600/ig/kg of actinomycin D. Microspherules (s)
are observed in the nucleolus. The specimen was fixed in glutaraldehyde and postfixed in osmium tetroxide. X 51,000.
123G
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Fio. 4. Same specimen as in Fig. 3, but fixation was carried out in osmium tetroxide and formalin. The chromatin is extracted, and
several dense microspherules (s) are apparent.
X 51,000.
FIG. 5. High magnification of the microspherules seen in the nucleoli of Ehrlich ascites cells treated with actinomycin D. The microspherules (s) contain dense fibrils. Some of them have a centrally located light area. The specimen was fixed in osmium tetroxide and
postfixed in formalin.
X 84,000.
JULY 1967
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Tadao Unuma and Harris Busch
Fio. 6. A nucléolosof an untreated Novikoff hepatoma cell. Chromatin (c) is evident within the nucleolus. The specimen was fixed
in glutaraldehyde and postfixed in osmium tetroxide.
X 29,000.
FIG. 7. The same specimen as in Fig. 6, but fixation was carried out in osmium tetroxide and formalin. Chromatin is extracted.
X 40,000.
FIG. 8. A nucleolus of a Xovikoff hepatoma cell, 60 minutes after treatment with 10 Mg/ml of actinomycin D in vitro. Microspherules
(s) are evident as are the halos around them. The microspherules are located almost exclusively at the periphery of this nucleolus. The
specimen was fixed in glutaraldehyde
and osmium tetroxide.
X 34,000.
1238
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VOL. 27
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FIG. 9. A micleolus of a Novikoff hepatoma cell, 69 minutes after treatment with actmomycui D of 890 Mg/kg of body weight in vivo.
The specimen was fixed in osmium tetroxide and formalin.
X 48,000.
FIG. 10. The same specimen as in Fig. 9, but fixation was carried out in formalin.
X 24,000.
FIG. 11. Pepsin treatment. The density of the microspherules (s) was reduced slightly. The granular and fibrillar components of the
nucleolus appeared more sharply after treatment. The specimen was the same as in Fig. 10. X 46,000.
FIG. 12. Ribonuclease treatment. The density of the microspherules was decreased remarkably. All the granular elements inside
the nucleolus disappeared. The perinucleolar chromâtin pattern was preserved. The specimen was the same as in Fig. 10. X 40,000.
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Tadao Unuma and Harris Busch
FIG. 13. Pepsin and ribonuclease treatment. The density of the microspherules (s) was decreased markedly compared with Pig. 11.
All the granular elements of the nucleolus and all the cytoplasmic ribosomes disappeared. The pepsin-resistant
matrix of the nucleolus
is visible. The specimen was the same as in Fig. 10. X 40,000.
FIG. 14. Deoxyribonuclease
treatment. The density of the microspherules (s) was not significantly decreased. The granular ele
ments of the nucleolus were intact but the nticleolus-associated
chromatin was removed. The specimen was the same as in Fig. 10.
X 43,000.
FIG. 15. Pepsin and deoxyribonuclease treatment. The density of the microspherules (s) was decreased slightly, compared with Fig.
14. The granular elements in the nucleolus appeared more sharply. No nucleolus-associated
chromatin was observed but the nuclear
ribonucleoprotein network in the nucleus was preserved. The specimen was the same as in Fig. 10. X 40,000.
FIG. 16. A nucleolus of an untreated Walker tumor cell. The nucleolus is composed mainly of granular components (g). Perinucleolar
chromatin with high electron density (c) is evident. The specimen was fixed in glutaraldehyde
and postfixed in osmium tetroxide.
X 34,000.
FIG. 17. A nucleolus of a Walker tumor cell, 60 minutes after treatment with 800 Mg/kg of actinomycin D. The nucleolus is compact.
The granular area (g) is decreased and is segregated sharply from the fibrillar area (f). The specimen was fixed in glutaraldehyde
and
postfixed in osmium tetroxide.
X 34,000.
FIG. 18. A nucleolus of a Walker tumor cell, (X)minutes after administration of 800 Mg/kg of actinomycin D. Vacuole-like areas (v)
are present. The specimen was fixed in glutaraldehyde and postfixed in osmium tetroxide. X 41,000.
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Microspherules
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Tadao Unuma and Harris Busch
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CANCER RESEARCH
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VOL. 27
Formation of Microspherules in Nucleoli of Tumor Cells Treated
with High Doses of Actinomycin D
Tadao Unuma and Harris Busch
Cancer Res 1967;27:1232-1242.
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