[CANCER RESEARCH 28, 81-90, January 1968]
Segregation
of the Nucleolus Produced
by Anthramycin1
Curtis Harris, Harold Grady, and Donald Svoboda
Department
oj Pathology, University oj Kansas Medical Center, Kansas City, Kansas 66103
SUMMARY
Anthramycin, an antibiotic possessing antitumor properties,
was administered to rats and mice in a single intraperitoneal
close ranging from 1 to 10 mg/kg body weight. Light micro
scopy showed that, at the highest dose, anthramycin produced
necrosis in the pancreas of rats but not in mice. By electron
microscopy, nucleolar changes were observed in the spleen and
kidney of both species by 1 hour. Acinar cells of the pancreas
showed nucleolar caps and segregation of nucleolar constituents
in both species at 3 to 4 hours. By 10 hours segregation of the
nucleolus and focal cytoplasmic degradation were prominent
in pancreatic cells of rats given higher doses but were not
observed in hepatocytes of either species.
Anthramycin produced nucleolar alterations in Kupffer cells,
endocrine and exocrine pancreatic cells, proximal tubular cells
of the kidney, and in the reticuloendothelial cells of the spleen
in rats and mice. The mechanism by which anthramycin acts
is unknown, but its ultrastructural effects, as well as its struc
tural similarity to actinomycin D, suggest that it may inhibit
RNA synthesis at the DNA level. The functional consequences
of segregation of the nucleolus have not been established.
INTRODUCTION
Recently, the effects of carcinogens and antitumor agents on
nucleolar morphology and function have been subjects of re
newed interest. Investigation of the acute ultrastructural and
biochemical abnormalities following administration of pyrrolizidine alkaloids (44), aflatoxin (42), ethionine (22), actinomycin
D (13, 15, 26, 35, 36, 41), 4-nitroquinoline-A^-oxide (34), UV
radiation (24), proflavin (38), and mitomycin (17) has indi
cated a variety of ultrastructural responses in the nucleus and
has emphasized the critical role of the nucleus in cellular
metabolism and inheritance. Particular emphasis has been
given to the nucleolus because of its importance in ribosomal
RNA (rRNA) synthesis and in the transfer of RNA from the
nucleus to the cytoplasm (27-30).
Anthramycin, an antibiotic isolated from a thermophilic
actinomycete (19), has in vitro antibacterial and in vivo antitumor activity (49). At concentrations effective as an antitumor agent against transplantable tumors in mice, anthrai This study was supported in part by USPHS Grants CA-08055
and CA-05680 and by the Kansas Division of The American Can
cer Society.
Received May 2, 1967; accepted September 8, 1967.
JANUARY
mycin does not produce toxicity in the bone marrow and
gastrointestinal tract (49). It possesses certain structural fea
tures in common with actinomycin D and the postulated biosynthetic intermediates of the actinomycin^, 3-hydroxy-4-methyIanthraniloyl peptides (3,47). Like kanamycin, neomycin, and
other antibiotics (48), anthramycin permanently bleaches
Euglena gracilis (11).
This report demonstrates that anthramycin is another agent
which causes nucleolar changes in pancreatic cells, reticulo
endothelial cells of liver and spleen, and in proximal tubular
epithelium in the kidney.
MATERIALS AND METHODS
Thirty-six inbred Fisher-344 male rats weighing between 95
and 240 gm and fifteen C57 black mice weighing between 20
and 30 gm were used. Anthramycin (anthramycin methyl
ether) dissolved in 100% ethanol was administered by a single
intraperitoneal injection. The dosages used were 1.0, 1.7, 5, and
10 mg/kg body weight. At least two rats at each dose were
sacrificed at 1, 4, 10, and 24 hr. Mice were sacrificed at 1, 3,
10, 12, and 24 hr.
Microscopic Studies. Small blocks of liver, pancreas, kidney,
and spleen were fixed in s-collidine-buffered 1% osmium
tetroxide for electron microscopy. The blocks were dehydrated
in a graded series of alcohols and embedded in Epon 812
containing 5% araldite (20). Thin sections were cut with an
LKB microtome using glass knives, stained with lead hydroxide
and/or uranium acetate, and examined in an RCA 3B or 3G
electron microscope. Semi-thin sections (0.5 to 1.5 /j.) of Eponembedded tissue were stained with aqueous azure A in an
equal volume of 5% sodium bicarbonate.
For light microscopy, portions of pancreas, liver, spleen,
kidney, and duodenum were fixed in formalin, embedded in
paraffin, and stained with hematoxylin and eosin or by the
periodic acid-Schiff procedure.
RESULTS
Light Microscopy
Loss of zymogen granules and necrosis were noted in the
pancreatic acinar cells of rats but not in mice. Necrosis of
duodenal crypts was present in both species. All pancreatic
alterations were most pronounced with the highest dose ( 10
mg/kg) at 10 hr. No changes were observed in the liver, kid
ney, or spleen.
1968
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81
C. Harris, H. Grady, and D. Svoboda
Electron Microscopy
Pancreas. At a dose of 5 mg/kg no changes were evident at
1 hr in the exocrine pancreas of rats. By 4 hr the endoplasmic
reticulum of acinar cells showed marked distention and vesiculation which was sustained at later intervals. The nucleoli of
acinar cells separated into granular and fibrillar components
(Figs. 1, 2) with a denser granular component occasionally
present at the periphery. It is uncertain whether these denser
granular components represent interchromatin granules, a
newly-formed element, or a degradation of préexistentele
ments. Nucleoli of islet cells were fragmented but no cytoplasmic changes were noted.
Nucleolar segregation was also apparent in many of the
nuclei at the lower dose (1 mg/kg) at 10 hr, and all nucleoli
examined were altered at the highest dose (10 mg/kg).
Granules similar in size to interchromatin granules were in
creased in number and usually clustered in the center of the
nucleus (Figs. 3, 4). Occasionally, the entire nucleoplasm ap
peared to be distinctly partitioned into two different densities
(Fig. 5). Focal cytoplasmic necrosis of exocrine cells was pres
ent (Fig. 6).
At 24 hr (1 mg/kg), most of the endocrine and exocrine cells
of the rat pancreas contained normal nucleoli but a round dense
nucleolar remnant consisting only of the fibrillar component
was present occasionally. Focal cytoplasmic necrosis persisted
in some exocrine cells.
The nucleolar response of mouse pancreas to anthramycin
was similar to that in rats. At 3 hr amidst acinar cells with
normal nucleoli, there were cells with dispersed and disarranged
nucleoli (Fig. 8). Nucleoli exhibited segregation at 4 and 12
hr (Figs 7, 9). The only cytoplasmic change was dilation of
the endoplasmic reticulum.
Liver. Segregation of the nucleolus was not observed in
either mouse or rat hepatocytes but granular aggregates meas
uring 0.2-0.4 /j. and nucleolar plaques were present (Fig. 10).
In both species, Kupffer cells showed nucleolar alterations at
all intervals (Fig. 11). No remarkable changes were observed
in the cytoplasm except the accumulation of fat droplets in
Golgi vesicles of rat hepatocytes at 24 hr.
Kidney and Spleen. Anthramycin produced a variety of
nucleolar changes in the kidney of rats. These alterations
ranged from fragmentation of the nucleolus to complete separa
tion of the nucleolar components (Fig. 12). In both species,
there were nucleolar alterations in reticuloendothelial cells of
the spleen (Figs. 13, 14).
DISCUSSION
Ultrastructure of the Normal Nucleus. The variation in
structure of normal nuclei of mammalian cells has been re
viewed by Davis (6), and attempts to unify the terminology
of the nucleolus have been reported at a recent symposium (46).
In general, the normal nucleolus is composed of (a) granules,
50-200 A in diameter; (o) fibrils, 50-100 A in diameter; and
(c) "intranucleolar chromatin," consisting of microfibrillar ele
ments (2). The first and second components are sensitive to
RNase digestion (21, 36) and the third component is partially
digestible by trypsin. Marinozzi and Bernhard (21) divided
82
the granular component into 2 types: one type resembles
cytoplasmic ribosomes 100-150 A in diameter and the second
consists of irregular aggregates 50-100 A in diameter. The
nucleolus is surrounded, in part, by the nucleolus-associated
chromatin which, like intranucleolar chromatin, contains DNA
(2, 45). The morphology of normal nuclei show some degree
of organ and species specificity. Nucleoli of rat and mouse
pancreatic cells are similar in their ultrastructure but nucleoli
of hepatocytes of several species of mice characteristically
show slight separation of the fibrillar and granular components.
Organ Specificity, Mechanism of Action, and Biochemical
Effects of Anthramycm. Nucleolar caps, produced by 4-nitroquinoline-A^-oxide, was first described by Reynolds et al. (34).
Like several other agents (39), anthramycin produces similar
changes in the nucleolus. In general, the initial separation of
nucleolar constituents is followed by a diminution of the
granular component.
The various agents which cause nucleolar segregation demon
strate organ specificity. Actinomycin D produces this nucleolar
change in the exocrine pancreas (13), cell culture (15), neurons
(16), and hepatocytes (26, 41). Anthramycin causes similar
abnormalities in nucleoli of islet cells, the exocrine pancreas
(Fig. 1), reticuloendothelial cells of the spleen (Figs. 13, 14),
and in Kupffer cells (Fig. 11), while hepatocytes show only
slightly altered nucleoli (Fig. 10). Aflatoxin (42), tannic acid
(31), and pyrrolizidine alkaloids (44) affect nucleoli of hepato
cytes and Kupffer cells but not those of the pancreas. Several
agents produce nucleolar segregation in cell culture ( 15, 17, 34,
35, 38). Two of the most unusual stimuli related to nucleolar
segregation are Mycoplasma (14) and Herpes simplex virus
(40), since these are the only known instances of nucleolar
segregation produced by microorganisms, though prominent
nucleolar changes of a different type occur in liver cells in
fected with Ectromelia (18). A separation of the entire nucleo
plasm into a lucent and a dense area was observed following
anthramycin treatment (Fig. 7). Actinomycin D (13) and proflavin (38) produce similar partition of electron density of the
nucleoplasm, and the same change has been observed follow
ing administration of cycloheximide (C. Harris, and D. J.
Svoboda, unpublished results).
The mechanism by which anthramycin acts is unknown. It
has been found to inhibit RNA synthesis in Ehrlich tumor
cells (G. Zbinden, personal communication). It possesses struc
tural features found in the actinomycins and their biosynthetic
intermediates (3, 47). Leimgruber et al. (19) suggested that
"anthramycin could be transformed in vivo to 'actinomycin
analogs', which actually may be responsible for the observed
anti-tumor activity."
Although the majority of agents which cause nucleolar segre
gation bind DNA and/or RNA (39), some DNA-alkylating
agents such as dimethylnitrosamine
(7), diethylnitrosamine
(23), and ethionine (22) do not cause the same nucleolar alter
ations. Work in progress in this laboratory indicates that, in
acute stages, dimethylnitrosamine, ethionine, and 3'-methyldimethylaminoazobenzene, when given in high doses, produce
a form of nucleolar segregation, suggesting that the nucleolar
effects of agents which alkylate nucleic acids are dependent
upon dose and time.
CANCER RESEARCH VOL. 28
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Segregation of the Nudeolus by Anthramydn
Although many of the nucleolar segregating agents are car
cinogens, there is no evidence implicating the related alterations
in nucleolar ultrastructure to carcinogenesis (43). Indeed, the
raethylation or ethylation of nucleotide bases, as with the
nitrosamines, may be meaningless in terms of transmission of
genetic information by DNA.
A recent comparative study of antimetabolites by Simard
and Bernhard (39) suggests that nucleolar segregating agents
". . . bind to DNA and interfere with its template activity for
the conduct of DNA-directed RNA synthesis by RNA polymerase." This hypothesis is compatible with the biochemical
mechanism of aflatoxin (9) and the proposed mechanism for
nucleolar caps induced by UV irradiation of the non-nucleolar
nucleoplasm (24). The recent finding in our laboratory that
cycloheximide can produce nucleolar segregation indicates that
an alteration in protein synthesis is an important facet of
nucleolar segregation (C. Harris and D. J. Svoboda, unpub
lished results). In this context, it is of interest that the primary
action of cycloheximide appears to be in the cytoplasm and the
drug interferes with protein synthesis at a step more distal in
the protein synthetic pathway than the site(s) of interference
or inhibition by other agents which cause nucleolar segregation.
The synthesis of rRNA seems to be particularly sensitive to
low concentrations of DNA-binding agents. As noted by Chiga
et al. (5), 80% of the nucleoli of hepatocytes undergo segrega
tion after administration of concentrations of actinomycin D
(13 /ig/100 gm body weight) which inhibit only a fractional
amount of DNA-dependent RNA synthesis. At a slightly higher
concentration of actinomycin D (20 ^g/100 gm body weight),
Jacob et al. (12) have indicated that the synthesis of the
initial rRNA species (45 S rRNA) is completely blocked within
30 seconds.
The protein matrix, of which 30% is histone, comprises ap
proximately 70% of the nucleolar dry weight (4, 10). Histones
appear to play an important role in RNA synthesis (1,4) and,
conceivably, changes in the structure or viscosity of the nu
cleolar protein matrix could produce secondary morphologic
alterations, such as segregation, in nucleoli. This consideration
seems all the more pertinent when one considers the stabilizing
effect of histones on nucleic acids (4) and the effects of cyclo
heximide on nuclear ultrastructure. Freedman et al. (8) found
that the inhibition of RNA synthesis by actinomycin D re
sulted in a 50% decrease in synthesis of the lysine-rich histone
fraction.
Anthramycin-induced changes parallel some of the morpho
logic effects of actinomycin D. Actinomycin D produces
nucleolar segregation and inhibits DNA-dependent RNA syn
thesis by binding the guanine residues of DNA (32, 33). As
suggested by Perry (29), actinomycin D would be expected to
inhibit the synthesis of high molecular weight guanine + cytosine-rich RNA, 45 S rRNA species, before low molecular weight
RNA, such as soluble RNA, would be inhibited. Indeed, the
45 S rRNA has been found to be sensitive to low concentra
tions of actinomycin D (12). The possibility that nucleolar
segregating agents, such as actinomycin D, may degrade or
alter newly synthesized nucleolar rRNA cannot be overlooked
(30). Schwartz and Garofalo (37) found little or no evidence
JANUARY
of migration of labeled 18 S and 28 S rRNA into the cytoplasm
following actinomycin D treatment.
Ultrastructural evidence indicates that anthramycin is a
typical nucleolar segregating agent which produces separation
of nucleolar constituents followed by a diminution of the
"granular component," 100-200 A in diameter. These nucleolar
granules and the 60 S ribosomal precursors observed in ultracentrifugation studies are most likely one and the same (25).
In conclusion, nucleolar segregation is the ultrastructural
reflection of nucleolar dysfunction, that is, alterations in the
synthesis of ribosomal precursors by various agents which bind
DNA and inhibit the synthesis of rRNA. Alterations in pre
existing nucleolar rRNA and protein synthesis may also be
partly responsible for the ultrastructural changes observed.
ACKNOWLEDGMENTS
We wish to thank Dr. Gerhard Zbinden, Research Division,
Hoffmann-La Roche, Nutley, New Jersey, for providing the anthra
mycin used in these studies. The technical assistance of Claire
Nielson, Kay Balle, Faye Brady, and Lynne Schmutz is gratefully
acknowledged.
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CANCER
RESEARCH
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VOL. 28
Segregation of the Nucleolus by Anthramycin
Figs. 1-14. All electron micrographs are from sections stained with uranyl acetate and lead.
Fig. 1. Rat pancreas, 4 hr after administration of anthramycin, 1 mg/kg body wt. The fibrillar component (/) and the granular com
ponent (g) show complete segregation. Ill-defined granular densities (arrows) are adjacent to the segregated nucleolus. X 72,000.
Fig. 2. Rat pancreas, 4 hr after administration of anthramycin, 1 mg/kg body wt. The granular component (g) is diminished and
segregated from the two contrasted zones of the fibrillar component (j¡and }t). A denser granular component (arrow) is present else
where in the nucleoplasm. X 66,000.
Fig. 3. Rat pancreas, 10 hr after administration of anthramycin, 5 mg/kg body wt. The nucleolus (nue) contains only the fibrillar
component and a constellation of granules (bracket) is centrally located. X 16,000.
Fig. 4. Rat pancreas, 10 hr after administration of anthramycin, 5 mg/kg body wt. The granular aggregate appears to be composed
of at least two different sizes of granules (1 and 2). X 61,000.
Fig. 5. Rat pancreas, 10 hr after administration of anthramycin, 5 mg/kg body wt. Distinct partition (marked by x) of the nucleo
plasm into regions of contrasting density is present. X 5,800.
Fig. 6. Rat pancreas 10 hr after administration of anthramycin, 5 mg/kg body wt. Foci of cytoplasmic necrosis containing mitochon
dria (m) and endoplasmic reticulum (ER) are prominent in acinar cells after high doses of anthramycin. X 6,700.
Fig. 7. Mouse pancreas, 4 hr after administration of anthramycin, 10 mg/kg body wt. The nucleolus is segregated into granular (g)
and fibrillar (/) components. The denser granular component (arrow) adjacent to the nucleolus is similar to those found in Figs. 3 and
4. X 29,000.
Fig. 8. Mouse pancreas, 3 hr after administration of anthramycin, 1.7 mg/kg body wt. The nucleolus shows fragmentation and partial
segregation of the granular (g) and fibrillar (/) components. X 39,000.
Fig. 9. Mouse pancreas, 12 hr after administration of anthramycin, 1 mg/kg body wt, Segregation of the fibrillar (/) and granular (g)
components of the nucleolus is present in the majority of the cells observed at 12 hr. X 19,000.
Fig. 10. Rat liver, 24 hr after administration of anthramycin, 1 mg/kg body wt. Hepatocytes show little response to anthramycin.
Nucleolar plaques (p) and granular aggregates (arrows) are noted with increased frequency. X 19,000.
Fig. 11. Rat liver, 24 hr after administration of anthramycin, 1 mg/kg body wt. Kupffer cell nucleoli demonstrate partial separation
of components at all intervals tested. X 29,000.
Fig. 12. Rat kidney, 12 hr after administration of anthramycin, 1 mg/kg body wt. Nucleolar alterations in the proximal tubule cells
are evident at all intervals beginning at 1 hr. Large microbodies (mb) containing dense nucleoids are seen. X 7,900.
Fig. 13. Rat spleen, 12 hr after administration of anthramycin, 1 mg/kg body wt. The fibrillar component (/) is partially encircled by
a ring of dense granules (g). X 72,000.
JANUARY
1968
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CANCER RESEARCH VOL. 28
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1968 American Association for Cancer Research.
Segregation oj the Nucleolus by Anthramycin
JANUARY 1968
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1968 American Association for Cancer Research.
C. Harris, H. Grady, and D. Svoboda
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CANCER
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1968 American Association for Cancer Research.
VOL. 28
Segregation of the Nucleolus Produced by Anthramycin
Curtis Harris, Harold Grady and Donald Svoboda
Cancer Res 1968;28:81-90.
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