Published June 1, 1967
LIGHT
AND
ELECTRON
RADIOAUTOGRAPHY
IN MICE
TREATED
J. C. H. D E
MICROSCOPIC
OF HEPATIC
WITH
MAN
CELL
ACTINOMYCIN
NUCLEOLI
D
and N. J. A. N O O R D U Y N
With the technical assistance of W. BEENS
From the Department of Submicroscopical Pathology, Pathologisch Laboratorium, University of
Leiden, Leiden, The Netherlands
ABSTRACT
Fine-structural alterations in nucleolar morphology as an effect of actinomycin D are well known.
They are described as a segregation of nucleolar
material into three components, i.e. granular,
fibrillar, and amorphous (1-4).
Apart from a general blocking effect on DNARNA transcription, low dosages of this drug are
known to have a rather selective inhibitory effect
on RNA synthesis in the nucleolus, as Perry (5)
showed in light microscopic radioautographic
studies.
Biochemically, Muramatsu et al. (6) found a
precursor-product relationship between 45, 35,
and 28S RNA's in the nucleolus of rat liver by
employing actinomycin D to suppress new RNA
synthesis in the nucleolus. They found a half-life
of 8 min for the 45S. A decrease of the radioactivity
of the newly synthesized 28S RNA was observed
after a 20 rain treatment with actinomycin D.
On the basis of these morphological and functional data, it seemed worthwhile to study the
effect of actinomycin D on the nucleolus by means
of high-resolution radioautography, with the
following questions in mind: (a) can a specific,
morphologically visible, fine-structural disturbance
exclusively located in the nucleolus be demonstrated in our material deriving from mice injected
intraperitoneally with actinomycin D? (b) is all
RNA labeling absent in the nucleolus when such a
disturbance becomes visibly manifest? (c) will the
489
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Nucleolar partition induced by actinomycin D was used to demonstrate some aspects of
nucleolar RNA synthesis and release in mouse hepatic cells, with light and electron microscopic radioautography. The effect of the drug on RNA synthesis and nucleolar morphology
was studied when actinomycin D treatment preceded labeling with tritiated orotic acid.
Nucleolar partition, consisting of a segegration into granular and fibrillar parts was visible
if a dosage of 25 #g of actinomycin D was used, but nucleolar RNA was still synthesized.
After a dosage of 400/~g of actinomycin D, nucleolar RNA synthesis was completely stopped
If labeling with tritiated orotic acid preceded treatment with 400 ~tg of actinomycin D,
labeled nucleolar RNA was present 15 min after actinomycin D treatment while high resolution radioautography showed an association of silver grains with the granular component.
At 30 rain after actinomicyn D treatment all labeling was lost, Since labeling was associated
with the granular component the progressive loss of label as a result of actinomycin D
treatment indicated a release of nucleolar granules. The correlation between this release
and the loss of 28S RNA from actinomyein D treated nucleoli as described in the literature
is discussed.
Published June 1, 1967
C
13.s -
C
13¸
C
10.2
C
7.8
6.5
N
N
i
N
2.3~
N
1.7~
lOO/~g
25¢/.g
200~g
C
o.2 Nr-1 0-5[-I
4oo/J.Cj
:ontr.
R N A release f r o m t h e a c t i n o m y c i n - t r e a t e d n u c l e o lus b e i m p a i r e d , or will t h e n u c l e o l u s still be able
to r e l e a s e R N A ?
MATERIAL
AND
METHODS
F e m a l e O20 mice weighing 20 g a n d aged a b o u t 2
m o n t h s were given a s t a n d a r d laboratory diet a n d
t a p water ad lib. All e x p e r i m e n t s were done at the
s a m e time of the day, between 11.00 a n d 12.00 a.m.,
in order to exclude d i u r n a l variation. Orotic acid5-3H (specific activity 2300-4000 m c / m m o l e , T h e
R a d i o c h e m i c a l Centre, A m e r s h a m , E n g l a n d ) was injected intraperitoneally in a dosage of 0.150 mc.
A c t i n o m y c i n D (kindly provided by M e r c k & Co.
Inc., R a h w a y , N e w Jersey) in saline was injected
intraperitoneally.
I n one set of experiments the a n i m a l s were injected
with a c t i n o m y c i n D in dosages of 25, 100, 200, a n d
400 # g 80 m i n before decapitation, a n d with orotic
acid-5-3H 20 rain before death.
I n a second set of e x p e r i m e n t s the a n i m a l s were
first injected w i t h orotic acid-5-3H, a n d 20 rain later
with 400 # g of a c t i n o m y c i n D. T h e y were sacrificed
15, 30, a n d 60 m i n after the a c t i n o m y c i n D t r e a t m e n t .
Control mice of t h e s a m e age a n d weight were injected with orotic acid-5-3H a n d sacrificed after 20
min. Tissue processing consisted of c u t t i n g small
(approximately 1 m m 3) blocks from the left lobe of
490
the liver, t h e n fixation for 1 hr in a m i x t u r e of two
parts o s m i u m tetroxide (2~o) a n d one p a r t collidine
buffer (7). After d e h y d r a t i o n in alcohol, the blocks
were e m b e d d e d in E p o n 812 (8). Sections 0.9 /z
thick were m a d e on a P o r t e r - B l u m M T - 2 m i c r o t o m e
a n d m o u n t e d on glass slides. For b o t h light a n d electron microscopic r a d i o a u t o g r a p h y , Ilford L-4 e m u l sions (10 g diluted in 20 ml distilled water) p r e p a r e d
by the loop m e t h o d according to Caro a n d v a n
T u b c r g e n (9) were used. T h i s type of emulsion was
especially suitable since the grain size is large e n o u g h
to be visible with the light microscope, a n d since in
electron microscopic r a d i o a u t o g r a p h y t h e emulsion
gives a localization w h i c h is sufficiently accurate for
t h e purposes dealt with in this paper. Silver grains
were c o u n t e d according to t h e m e t h o d already described in a previous p a p e r (10).
F r o m s o m e of t h e liver samples, u l t r a t h i n sections
were m a d e for high-resolution r a d i o a u t o g r a p h y .
Electron microscopic r a d i o a u t o g r a p h y was done
with Ilford L-4 emulsion p r e p a r e d according to
m e t h o d s t h a t have already been reported (10, 11).
I n addition, K o d a k N T E emulsion was p r e p a r e d
according to a m e t h o d described by Salpeter a n d
B a c h m a n n (12), a n d t h e sensitivity of this emulsion
was e n h a n c e d with t h e gold latensification m e t h o d
as described by these a u t h o r s (12). After exposure
a n d d e v e l o p m e n t of r a d i o a u t o g r a p h s , u l t r a t h i n seetions were stained with lead cacodylate (13).
THE JOURNAL OF CELL BIOLOGY • VOLUME 33, 1967
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FmTJRE 1. Grain counts in nucleus (N) a n d cytoplasm (C) of 30 randomly chosen liver cells of mice
treated for 80 rain with varying doses of actinomycin D. Labeling with orotic acid-5-~H was started ~0
rain before death.
Published June 1, 1967
of actinomycin D after initial labeling for 20 min. Some partition is already visible (broken line), f, fibrillar component. There is an indication of fibrils (arrow). Silver grains occur over granular part (g).
X 50,000.
RESULTS
To establish the effect of different dosages of
actinomycin D on the RNA synthesis in the liver
cell, the following experiment was done.
The effect of actinomycin D treatment at 60
min before labeling with orotic acid-5-3H on the
incorporation of the tritiated orotic acid into RNA
as measured by grain counting in radioautographs
of 0.9 ~ sections is shown in Fig. 1. For comparison,
normal values are included in the graph. It will
be seen that a dosage of 25 /~g of actinomycin D
decreases the number of grains in the nucleus,
whereas the number of grains in the cytoplasm is
not significandy changed. With higher dosages of
100 and 200 gg the number of grains in both the
nucleus and the cytoplasm decreases progressively,
and with the highest dosage used the number of
grains in the nucleus and the cytoplasm becomes
negligible.
Fine-structural changes induced by actinomycin
D are especially prominent in the structure of the
nucleolus. Normally, the nucleolus shows a folded
structure, the nucleolonema, consisting of finely
granular and fibrillar electron-opaque components
through which numerous, larger (about 180 A)
granules with the appearance of ribosomes are
randomly scattered. In high-resolution radioautographs of normal nucleoli the silver grains are
randomly scattered over the nucleolus (Fig. 4). In
liver cells of animals treated with actinomycin D,
however, there is a striking demarcation between
the finely fibrillar component and a coarsely
granular ribosome-like component, the latter being
distributed in crescents at the periphery of the
structure. After prolonged treatment with higher
dosages of actinomycin D (Figs. 9 and 10), these
changes are more conspicuous. Strongly electronopaque clusters of coarsely granular material are
then visible at the outer boundary of the nucleolus.
In many instances, loosely arranged agglomera-
J. C. H. DE MAN AND N. J. A. ~'OORDUYN Hepatic Cell Nucleoli Treated with Actinomycin D
491
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FIGURE 2. High-resolution radioautograph of nucleolus of mouse liver cell treated for 15 rain with 400 #g
Published June 1, 1967
tions of ribosome-like particles are found in the
s u r r o u n i n g d nucleus. These agglomerations a p p e a r
to be connected with the g r a n u l a r c o m p o n e n t of
the nucleolus. A l t h o u g h these alterations were
especially m a r k e d after the use of higher dosages of
actinomycin D, partition was also visible to some
extent w h e n a dosage of 25 ~g was used for 80 m i n
(Fig. 6). T h e r a d i o a u t o g r a p h s in this latter instance were interesting because they showed t h a t
a l t h o u g h radioactivity was very low in these
nucleoli a few grains were occasionally found (Figs.
5 a n d 6). These grains were located in the g r a n u l a r
p a r t of the nucleolus (Fig. 6), thus indicating t h a t
with a dosage of 25 # g some R N A labeling still
occurs in spite of prolonged (80 min) t r e a t m e n t
with a c t i n o m y c i n D.
492
T h e second set of experiments was designed with
the purpose of establishing the effect of actinomycin D on the release of labeled R N A from the
nucleolus. Therefore, actinomycin D t r e a t m e n t
was initiated after a previous labeling with orotic
acid-5-3H for 20 rain. Actinomycin D was used in
a dosage of 400 #g, since the preceding experiments
h a d shown that with this dosage R N A synthesis is
virtually blocked. Figures 7 a n d 8 show t h a t at 15
rain after t r e a t m e n t with actinomycin D several
silver grains occur over the nucleoli, whereas at 30
m i n after t r e a t m e n t labeling is absent. This indicates that labeled R N A is lost from the nucleolus.
C o n t r a r y to this loss of nucleolar labeling, silver
grains are still present over other parts of the
nucleus a n d the cytoplasm. High-resolution
TNE JOURNAL OF CELL BIOLOGY • VOLUME33, 1967
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FIGURE 3. Same as Fig. 2. Kodak NTE emulsion with gold latensification instead of Ilford L-4 emulsion
as in the other electronmicroscopic radioautographs. )< 87,000.
Published June 1, 1967
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FIGU~ 4. High-resolution radioautograph of normal nucleolus. Labeling time, 20 rain. X 40,000.
radioautographs of the liver cells treated with
actinomycin D for 15 min are interesting because
they show that silver grains are associated with the
coarsely granular components of the nucleolus
(Figs. 2 and 3).
DISCUSSION
In general, the actinomycin D-induced alterations
of the nucleolus agree well with the extensive
descriptions in the literature, although an amorphous component was infrequently found. In our
material the extent of the changes shows a dosedependency. It is interesting that the minimal
structural changes seen in nucleoli of animals
treated with 25 #g of actinomycin D seem to be
compatible with some R N A labeling in the nucleolus (Figs. 5 and 6). It is also clear that if a high
dosage (400/zg) is given the extent of the changes
increases with the duration of treatment.
Whereas in the normal nucleolus the ribosomelike particles are randomly distributed throughout
the structure, as a result of actinomycin D treatment the ribosome-like particles are moved to the
periphery of the nucleolus. The occurrence of
loosely packed agglomerations of ribosome-like
particles in the surrounding nucleus, which are,
however, continuous with the granular part of the
nucleolus proper (Figs. 9 and 10), even seem to
suggest a release of such particles. This phenomenon runs parallel with the finding of a loss of
J, C. I~I. DE MAN AND N. J'. A. NOOI~DUYN Hepatic Cell Nucleoli Treated with Actinomycin D
493
Published June 1, 1967
FIGURES 7 and 8. Light microscopical radioautographs of mice treated for 15 min (Fig. 7) and 30 min
(Fig. 8) with 400 #g of actinomycin D after an initial labeling for ~0 rain. Some of the silver grains in Fig.
7 are associated with the nucieoli (arrow). No such association of silver grains with the nucleoli is found
in Fig. 8. Figs. 7 and 8, X le00.
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FIGURES 5 and 6. Light microscopical (Fig. 5) and high-resolution (Fig. 6) radioautographs of mouse
liver cells treated for 80 rain with ~5 #g of actinomycin D. Labeling started ~0 rain before death. Silver
grains in Fig. 5 ale shown to be occasionally associated with nucleoli (arrow). Figure 6 shows the granular component being shifted to the periphery of the nucleolus. Silver grain is associated with granular
part. Fig. 5, X le00; Fig. 6, X ~°1,000.
Published June 1, 1967
labeling of the nucleolus between 15 a n d 30 m i n
after t r e a t m e n t with 400 gg of a c t i n o m y c i n D. T h e
loss of labeling is p r o b a b l y related to the release of
the ribosome-like particles, since in Figs. 2, 3 a n d
6 it can be seen t h a t the silver grains are associated
with the g r a n u l a r p a r t of the actinomycin-D
treated nucleolus. This assumption is further supported by the work of M u r a m a t s u et al. (6). They
found a decrease of radioactivity of newly synthesized 28S R N A in the nucleolus between 20 and 40
m i n after actinomycin D treatment. Since 28S
R N A is associated with 60S ribosomal particles, it
seems justified to consider the nucleolar granules
as 60S ribosomal particles. It m a y be assumed that
at 15 m i n after a high dose of actinomycin D which
stops all new synthesis of R N A the conversion of
precursor R N A to 28S R N A which runs parallel
with a migration of radioactivity from fibrillar to
g r a n u l a r m a t e r i a l (14) can be expected to ke
practically completed since according to M u r a matsu (6) the half-life of precursor R N A is a b o u t
8 min. Geuskens' finding (3) of an impaired release
of newly synthesized R N A f r o m actinomycin
D-treated nucleoli in cultured monkey kidney cells
m a y be a t t r i b u t e d to differences in the experimental material a n d techniques used.
This research was supported by the Queen Wilhelmina Foundation against Cancer.
Receivedfor publication 26 September 1966.
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J. C. H. DE MAN AND N. J. A. NOORDUYN Hepatic Cell Nudeoli Treated with Aetinomycin D
495
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FIGURES 9 and 10. Electron mierographs of nucleoli of mouse hepatic cells treated for 30 min (Fig. 9)
and 60 rain (Fig. 10) with 400 #g of actinomycin D. Both micrographs show that the nucleoli have a
centrally located fibrillar component and a peripheral granular part. Note continuity between the loosely
arranged agglomerations of ribosome-like particles in the surrounding nucleus and the granular component of the nucleolus. Figs. 9 and 10, X ~1,600.
Published June 1, 1967
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THE JOURNAL OF CELL BIOLOGY • VOLUME 33, 1967