[CANCER RESEARCH 29, 1875-1880, October 1969]
Electron Microscopic Studies on HeLa Cells Exposed
to the Antibiotic Toyocamycin
Ursula Heine
Viral Biology Branch, National Cancer Institute, Bethesda, Maryland 20014
SUMMARY
The antibiotic toyocamycin (TCM) selectively inhibits the
synthesis of ribosomal RNA in different mammalian cells by
suppressing the cleavage of the 45 S RNA precursor to 18-28 S
RNA (27). Present ultramorphologic studies on HeLa cells
exposed to TCM reveal a highly specific response to the drug.
When employed in low concentrations, TCM selectively
induces an enlargement of the pars fibrosa in the nucleolus of
the treated cells. High doses of TCM, however, provoke
morphologic changes similar to those observed with the action
of antibiotics interfering with the template activity of DNA.
INTRODUCTION
It was recently found that the antibiotic toyocamycin (TCM)
selectively inhibits ribosomal RNA (rRNA) synthesis in HeLa
cells, mouse fibroblasts, and Ehrlich ascites cells (27). At low
concentrations, the drug exclusively suppresses the formation
of 28 S and 18 S RNA in the cell systems studied, but at the
same low concentration it does not affect the synthesis of
32-45 S RNA, 4 S RNA, DNA, or protein. According to the
biochemical data published, it is preponderantly the 45 S RNA
which accumulates in the nucleolus of the treated cells. It was
shown that toyocamycin [4-amino-5-cyano-7-/3-D-ribofuranosyl-pyrrolo (2,3,-d)pyrimidine] (TCM) is at least partly
incorporated into this 45 S RNA molecule, most probably
replacing adenosine (27).
Previous investigations have emphasized the importance of
the nucleolus in cellular metabolism, i.e., in the synthesis of
rRNA, it is known that a nucleolar ~ / A with a sedimentation
coefficient of 45 S is the precursor of rRNA (15, 16, 17, 18,
22). There is evidence that a distinct morphologic entity, a
fibrillar component of the nucleolus, might represent the 45 S
precursor of rRNA (see Refs. 2, 8). The high specificity of
TCM, in selectively inhibiting the maturation of this precursor,
provides an excellent tool for attempting to correlate the
biochemical and morphologic parameters of this entity.
For these reasons, it is of much interest to study the highly
selective influence of TCM on cellular fine structure, especially
that of the nucleus and nucleolus, and to investigate further
Received December 31, 1968; accepted June 24, 1969.
the 45 S component which presumably accumulates in the
nucleolus in the presence of the drug.
This report demonstrates that changes in cellular ultramorphology are very specific when TCM is employed in low
concentrations, whereas high concentrations of the same
antibiotic produce nuclear changes comparable to those
observed under the influence of actinomycin D.
MATERIALS AND METHODS
Cells and Media. In order to facilitate a comparison of
biochemical data already compiled with the morphology to be
studied, our experiments were designed to be as similar as
possible to those already published (27).
tteLa-S a cells used in this study were obtained either from
Flow Laboratories, Inc., Rockville, Maryland, or from the
Tissue and Media Unit of the NIH, Bethesda, Maryland. The
cells were grown in Erlenmeyer flasks as suspension cultures at
a concentration of 5-6 X 105 cells/ml. The medium used was
Eagle's minimum essential medium (4), supplemented with 10%
heat-inactivated calf serum, 3% glutamine, penicillin (100
units/rnl), and streptomycin (100 tag/ml). Cultures were
routinely checked for contamination with mycoplasma and
were found to be negative.
Antibiotic. TCM was a generous gift from Dr. Suhadolnik,
Albert Einstein Medical Center, Philadelphia, Pennsylvania. It
was added to the tissue culture medium in concentrations of
0.02, 0.01, 1.0, or 20.0/ag/ml. The cells were exposed to the
drug for different lengths of time, ranging from 1 hr to 2 days,
depending on the concentration used in the different
experiments.
Fixation and Embedding. Cells were fixed in the cold in
suspension by diluting cultures with equal volumes of 3%
glutaraldehyde buffered to pH 7.45 with sodium cacodylate
(21). The fixation time was 15 min, after which the
suspensions were pelleted at 2000 rpm in an International
PR-2 refrigerated centrifuge. The pellets were postfixed with
Dalton's chrome-osmium fixative (3), and embedded in
Epon-Araldite (14). Ultrathin sections were cut on an
LKB-Ultrotome, transferred to Formvar-coated grids, and
stained with uranyl-acetate (28) and lead citrate (19). They
were examined in a Siemens Elmiskop I with double condenser
and 50/a objective aperture. An accelerating voltage of 80 kv
was used.
OCTOBER 1969
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1875
Ursula Heine
RESULTS
Controls
In the controls a large nucleus is surrounded by a small rim
of cytoplasm. The nuclear membrane has many invaginations.
Mitochondria and free ribosomes, mono- and polysomes, are
numerous, whereas lysosomes and rough endoplasmic reticulum are sparse. In the interphase nucleus the major part of the
cbaomatin is distributed evenly (diffuse chromatin). Only
small patches of condensed chromatin are arranged adjacent to
the nuclear membrane and throughout the nucleolus. Perichromatinic granules are frequent. The nucleolus is large, with its
granular component (pars granulosa) being outstanding (Fig. 1,
A). Areas of fine fibrillar material are present in it (Fig. 1, C).
These areas are surrounded by the fibrillar component (pars
fibrosa) of the nucleolus (Fig. I, B), which contributes very
little to the whole structure.
Response to TCM in Low Concentration
0.01 ~g TCM/ml. The influence of the drug on ultrastructural
changes in the cells is minute when applied in this low
concentration. Nucleoli appear to be slightly larger than those
in controls, and a small increase in fibrillar material has been
observed after exposure to the antibiotic for 2 days.
0.1 pg TCM/ml. First changes in fine structure are evident
only in the nucleoli after exposure to the antibiotic for 1 hr.
As shown in Fig. 2, they are expressed by a loss of many of
the nucleolar granules. The remaining nucleoli are mainly
composed of fibrillar material, arranged into irregularly intertwined cords (nucleolonema) with few granules in their
immediate proximity. Further and striking changes in fine
structure are seen after exposure to the antibiotic for from 6
to 12 hr. The organelles are often round and exhibit a
pronounced density due to a substantial increase of their
fibrillar component, paralleled by a diminution of their
granular part (Fig. 3). The overall size of these nucleoli is
similar to that seen in the controls. This process, the
accumulation of fibrils, accompanied by a loss of granules,
continues during the following 12 hr and results finally in
large, round nucleoli of high density.
1.0/ag TCM/ml. The ultracytologic response of the cells to
this concentration is similar to that provoked by 0.1 /ag
TCM/ml. However, the spherulization of the nucleolus and the
accumulation of the fibrillar component in it are already
pronounced in less than 6 hr (Fig. 4).
Response to TCM in High Concentration
20/ag TCM/ml. Early changes, after 1 to 6 hr exposure,
predominantly effect the nucleus. These changes are similar to
those caused by other antibiotics and drugs, such as
actinomycin D (9, 11,13), mitomycin C (13), anthramycin (7),
aflatoxin (1), and 4-nitroquinolin4V-oxide (20), and they are
expressed by a "nucleolar segregation" (25), i.e., the
separation and redistribution of amorphous, fibrillar, and
granular components into distinct entities (Figs. 5, 6).
1876
Contrary to the action of the drugs just mentioned,
especially that of actinomycin D, large masses of the granular
component persist in the nuclei during an extended exposure
(up to 24 hr) to TCM. They are still present when the
clumping of chromatin is already evident (Fig. 7), and cell
death occurs.
Dense granules of unknown origin with an average diameter
of 200-300 m~ are another characteristic feature of the nuclei
in these later states. They form small accumulations near the
nucleolar masses and are also distributed singularly throughout
the remaining nucleoplasm (Fig. 7).
The late nuclear changes are paralleled by deviations from
the normal in the cytoplasm of the cells. These deviations are
an increase in the number of lysosomes, the breakdown of
polysomes to monosomes, and the extrusion of small blebs of
cytoplasm into the medium resulting in a gradual loss of a
major part of the cytoplasm in the affected cells.
DISCUSSION
It is now well established that 45 S to 32 S rRNA precursors
are formed in the nucleolus of the cells (15-18, 22). Kinetic
studies give evidence that a substantial amount of the
precursor can be detected in HeLa cells as early as 30 min after
labeling with 32 p, whereas a label applied for a shorter period
of 5 min can only be recovered in an RNA of great
heterogeneity of size (10-100 S), which is not of the ribosomal
type (10). As shown, in the presence of 0.1 /ag TCM/ml for 6
hr, the 45 S rRNA accumulation in mammalian cell nucleoli is
about 4-fold. At the same time, its maturation to 28 S and 18
S RNA is completely interrupted. There is no effect on the 4 S
RNA (27).
The changes in nucleolar fine structure presented here after
exposure to the same doses of TCM (0.1 tag]ml)for a
prolonged time, 12 to 24 hr, result in a profound increase of
material resembling that of the pars fibrosa. In agreement with
the biochemical data mentioned above, it might be assumed
that this material contains the 45 S rRNA. The small amount
of granules which remain recogn~able between the electron
dense fibrils might then represent the components containing
35-32 S RNA. According to the biochemical data the presence
of 28 S RNA can be excluded (27).
A short exposure to 0.1 ~g TCM/ml for 1 hr results in an
obvious loss of nucleolar granules. The remaining nucleolus is
represented by a twisted and skein-like structure which bears
some resemblance to forms observed during "reconstruction"
of hepatic nucleoli after treatment with ethionine that was
followed by methionine. Those structures were designated so
as to be fundamental units in the restoration of the nucleolus
(24). The pronounced appearance of similar structures early in
our experiments might be due to (a) the transfer of ribosomal
components already present at the start of the experiment into
other areas of the nucleus and into the cytoplasm, and (b) the
relatively small amount of ribosomal precursors manufactured
during the short exposure to TCM.
The results of this work find support in earlier publications
discussing autoradiographic studies on nucleoli of different
cells (5, 6). The Chironomus salivary gland cell, e.g., accumulates label-designating rRNA precursor in the nucleolus over
CANCER RESEARCH VOL. 29
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HeLa Cells and T o y o c a m y c i n
the pars fibrosa after exposure for 13 min. The label shifts
later to the granular part of the same organelle and can be
found there as late as 46 hr after application (6). In addition,
recent investigations on cells under thermic shock led to the
belief that the fibrillar component of the nucleolus might
constitute the 45 S ribosomal precursor (26).
However, the correlation of the granular component of the
,~ucl~.olus with an RNA of specific sedimentation velocity is
still under discussion. The autoradiographic studies mentioned
above are comparable with the conclusion that 35 S and
possibly 28 S rRNA, but little of the 45 S precursors are
located in the granules. The effect of actinomycin D on cells
of different types supports this assumption (18).
Although we could only detect traces of granular material in
nucleoli under the influence of small doses of TCM, the
accumulation and persistence of the nucleolar granules after
exposure to high doses of the drug remains a puzzling
problem. A possible explanation might be found by considering
changes in the micro environment of the nucleolus, due to the
conditions of the experiment, as a decisive factor in the
formation of the granules. As shown in this report, high doses
of TCM not only influence the morphology of the nucleolus,
but are also related to severe changes in the other parts of the
nucleus including changes in the physical state of chromatin
(redistribution and clumping). This holds true for the action of
other drugs, such as actinomycin D (9, 11, 23), anthramycin
(7), and aflatoxin (1), which were studied previously, and for
the response to an infection with mycoplasma (12, see also
Ref. 25). It might be feasible to speculate that these
environmental changes influence much of the 45 S rRNA
precursor which accumulates in the presence of TCM and
promote its cleavage to the 32-35 S component, a process
which would have its morphologic manifestation in the
maturation of fibrils to granules.
The observed persistence of the granules, until cell death
under the influence of TCM, is in good agreement with the
biochemical findings (27) indicating that once the drug is
incorporated into the precursor molecule, cleavage into 28 S
and 18 S RNA-containing substructures is not possible.
Previous biochemical and present morphologic studies concerning the action of the antibiotic TCM on cellular metabolism open interesting aspects for further investigations in
analyzing DNA-dependent RNA synthesis as a nucleotar
function.
ACKNOWLEDGMENTS
The author wishes to express her gratitude to Dr. Josef R~man,
Institute of Organic Chemistry and Biochenfistry, Czechoslovak
Academy of Science, Prague, Czechoslovakia, for many helpful suggestions and criticisms throughout this work, and to Dr. A. J. Dalton for
advice in the preparation of the manuscript. The valuable technical
assistance of Mr. R. Moore and Mr. R. Steinberg is gratefully
acknowledged.
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Figs. 1-7. All specimens were fixed with glutaraldehyde, postfixed with chrome-osmium, embedded in Epon-Araldite, and stained with uranyl
acetate and lead citrate.
Fig. 1. HeLa cell, control. Nucleolus with abundant granules, pars granulosa (A), small areas of fibrils, pars fibrosa (B), and two areas containing
material of very fine fibers (C). Chromatin (CH); nuclear membrane (NM).X 30,000.
Fig. 2. HeLa cell exposed to 0.1 pg toyocamycin/mI for 1 hr. The loss of nucleolar granules (/1) is evident. The nucleolus consists mainly of cords
of fibrillar material, nucleolonema (B), with few ribosome-like granules in their immediate proximity. Two structures (C) similar to those
designated as C in Fig. 1 are recognizable. X 30,000.
Fig. 3. Nucleolus of a HeLa cell after treatment with 0.1 pg toyocamycin/ml for 12 hr. The nucleolus is very dense. The areas designated as C in
the control are still recognizable (C). The distinction between fibrils and granules is difficult due to the extreme density of the structure. The
nucleolus contains many fibrils (B), and few granules (A) are distributed at random throughout the structure. X 30,000.
Fig. 4. HeLa cell exposed to 1 btg toyocamycin/ml for 6 hr. The nucleolus forms a large, dense body. Its major component is fibrils (pars fibrosa).
Few granules, remnants of the pars granulosa, with a diameter of approximately 150 A can be recognized throughout the structure (see inset
arrow). X 30,000. Inset, X 120,000.
Fig. 5. Nucleus of a HeLa cell after treatment with 20 pg toyocamycin/ml for 2 hr. The observed alterations affect only the nucleolus. The
separation of its components is evident resulting in few accumulations of granules (,4); areas of high density containing the fibrillar component (B)
also result. Chromatin is distributed diffusely throughout the nucleus. X 18,000.
Fig. 6. Nucleolus of a HeLa cell treated in a similar manner as illustrated in Fig. 5, but at a higher magnification. The separation of the nucleolar
components is very pronounced. The granular part of the nucleolus is large and contained in the center of the photograph (A). Fibrillar part of the
nucleolus (B). X 32,000
Fig. 7. Nucleolus of a HeLa cell exposed to 20pg toyocamycin/ml for 24 hr. After prolonged exposure to TCM in high concentration, the visible
alterations affect the nucleolus as well as other parts of the nucleus. A nucleolar segregation is evident. The granular part of the nucleolus is
prominent (A), and a small area of fibrils is recognizable (B). Chromatin (CH) is clumped and accumulated adjacent to the nuclear membrane.
Dense granules (arrows), about 200-300 mp in diameter, are found in clumps adjacent to the granular component of the nucleolus. Individual
electron-dense granules are also distributed throughout the remaining nucleoplasm. X 17,500.
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C A N C E R R E S E A R C H V O L . 29
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OCTOBER 1969
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CANCER RESEARCH VOL. 29
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Research.
Electron Microscopic Studies on HeLa Cells Exposed to the
Antibiotic Toyocamycin
Ursula Heine
Cancer Res 1969;29:1875-1880.
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