Download Electron Microscope Studies on HeLa Cell Lines

Electron Microscope Studies on HeLa Cell Lines
Sensitive and Resistant to Actinomycin D*
L. J.
JOURNEY AND MILTON N . GOLDSTEIN
(Department of Experimental Biology, Roswell Park Memorial Institute, Buffalo, New York)
SUMS{ARY
The fine structure of stock IIeLa cells has been examined, and several new details
have been described. Certain specific structural alterations induced by actinomycin D,
a cytotoxic antibiotic, have been noted. These alterations included cytoplasmic blebbing and a fragmentation of the nucleoli accompanied by a pronounced loss in their osmiophilia, tteLa cells made resistant to actinomycin D did not display these structural
changes. The findings are discussed in the light of histochemical and biochemical evidence which indicates that actinomycin D inhibits RNA and protein synthesis.
Actinomycin D, an antibiotic isolated by Manaker et al. (19) from Streptomyces parvallus, has
caused regression of some animal and human tumors ( l l , 22, 25). This antibiotic is toxic in vivo,
and cytotoxic effects are produced in cells of newly
explanted and established cell lines grown in vitro
(4, 6, 7, 12).
Recently, we reported some cytological, histochemical, and chemical studies on a stock HeLa
line which was sensitive to actinomycin D and a
resistant line isolated from the stock HeLa cells
(15). The drug produced alterations in nucleolar
structure and a decrease in staining intensity for
R N A in nucleoli and cytoplasm of sensitive cells,
whereas the resistant cells were not affected.
Chemical assays revealed a decrease in R N A and
protein, whereas D N A was unchanged in treated,
sensitive cells.
The present report describes more detailed
phase and electron microscopic observations on
sensitive HeLa cells treated with actinomycin D
and a HeLa line made resistant to the drug.
199. Details of culture technics and development
of the resistant line are described elsewhere (]5).
The experimental material included three cell
strains:
1. HeLa-S--stock cell line that was sensitive
to actinomycin D.
2. H e L a - S R - - a partially resistant sublinc selected from the above stock culture by intermittent exposure to actinomycin D. These cells
survived for longer than 10 days in 0.1 #g/ml
actinomycin D, a concentration lethal for sensitive
cells within 72 hours.
S. H e L a - R - - a resistant subline that can actively multiply in medium containing 0.1 pg/ml of the
antibiotic.
For E M studies the cells were explanted into
15 X 150 ram. :Pyrex test tubes containing 1.5 ml.
of medium. Cultures were refed with experimental
medium 24 hours after explantation. At the end of
the experimental period (24-48 hours), controls
and drug-exposed cells were washed in Hanks
solution and fixed for 10 minutes in osmium, vapor.
The cells were then rapidly dehydrated in a
graded series of alcohols. Final embedding was
M A T E R I A L S AND M E T H O D S
done in pre-polymerized methacrylate (4 butyl-IThe HeLa lines were derived from stock HeLa 1 methyl) and polymerization completed at
cells that have been serially propagated in our 70~ C. Embedded cells were removed by cracking
laboratory for the past 5 years in a medium com- the tubes in a freezing mixture of dry ice and alcoposed of 1 part calf serum and 2 parts of medium hol. Selected areas were cut out, oriented, cemented to Lucite rods, and sectioned in a :Porter* Supported in part by research grant t/Dl~G-527 from tlle Blum microtome. The sections were picked up on
Damon Runyon Memorial Fund for Cancer l~esearch.
Formvar-coated grids and examined in an RCA
Received for publication March 9, 1.q6l.
EMU-2B microscope.
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Cancer Research
RESULTS
Stock HeLa.--When explanted in roller tubes,
cells adhered to the glass and grew out in dense
monolayers. Cell surfaces had numerous villi,
which interdigitated with surrounding cells (Figs.
3 and 7). In certain areas cell surfaces were in
close apposition, but in other portions of the sheet
cells were separated by wide intercellular spaces.
Typically, each control cell possessed a large
ovoid nucleus, though in some dense areas multinucleated cells were apparent. Some nuclei had
deep invaginations, and others had bhmt protrusions or lobulations. The nucleoplasm was composed of fine, granular material distributed homogeneously. Chromatin material was evenly disperscd throughout the nucleoplasm with no
margination along the nuclear envelope. All stages
of mitosis wcre cncountered; chromosomes appeared as condensed masses of densely staining
granules, and the filamentous structure of the
spindle apparatus was evident.
Nuclei contained one to five nucleoli, which
varied considerably in size, and many were found
adjacent to the nuclear envelope. Some nucleolar
structures appeared as profiles of coiled threadlike
nucleolonema (Fig. 7), and a few contained dense
inclusions. At higher magnifications, nucleoli presented the typical picture of osmiophilic granules
resembing the ribonucleoprotein particles of the
cytoplasm.
Mitochondria were extremely pleomorphic,
ranging from small, round types to huge elongate
varieties, which may branch considerably to produce bizarre shapes (Fig. 3). The arrangement of
internal cristae was variable. Septation was transverse, oblique, or longitudinal with respect to the
long axis of the mitochondrion. Golgi material was
usually found near the nucleus and consisted of a
complex network of membranes with associated
vacuoles and granules (Fig. 7). Dense bodies,
smaller than mitochondria and with no internal
structure, were also present (Fig. 7). These structures resembled the "globoid bodies" found in
HeLa cells by Harford et al. (16). Similar granules
have been described in other tissues and have been
termed "microbodies" (24), "peribiliary bodies"
(21), "dense bodies" (20), and apparently correspond to the "lysosome" fraction obtained from
tissue homogenates (5).
Organized cndoplasmic reticulum was sparse in
HeLa cells, with little evidence of the orderly
array of tubules and sacs seen in many normal
tissues. This systcm was represented predominantly as isolated vesicles and tubules dispersed
throughout thc cytoplasm. Other cytoplasmic
structures found occasionally were areas composed
Vol. ~1, A u g u s t 1961
of fibrous material. In many cells, certain areas of
the cytoplasm appeared hyaline or less osmiophilic
than surrounding portions (Fig. 7). Although
sharply demarcated, these areas were not membrane-enclosed and contained the same granular
background as adjacent portions of the cytoplasm.
HeLa-S + Actinomycin D.--After exposure to
1 gg/ml of actinomycin D for 20 hours, extensive
cytotoxic alterations were evident in sensitive
HeLa cells. Some cells showed cytoplasmic
blebbing, i.e., out-pocketing and pinching-off of
cytoplasmic contents. This process is demonstrated in Figure 2, a phase-contrast picture of a
living HeLa cell with numerous surface blebs. A
HeLa cell in control medium (Fig. 1) is included
for comparison. Successive stages in the formation
and release of cytoplasmic blebs are seen in greater
detail with electron microscopy (Fig. 6). Other
alterations in fine structure inchlded vacuolization
of mitochondria and crgastoplasmic vesicles, and
disruption of nuclear contents.
Nucleoli were especially sensitive to the cytotoxic effects of actinomycin D. They were converted from densely stained bodies to nucleolar
structures composed of a hyaline, washed-out
interior enclosed in a peripheral ring of osmiophilic granules. Actinomycin D also induced a
marked budding and fragmentation of the nucleoli
(Figs. 5 and 6). I t should bc emphasized that
nucleolar alterations were apparent in all of tile
cells examined.
HeLa-SR.--The following observations were
made on a subline intermittently exposed to
actinomycin D and fixed for electron microscopic
examination after 24 hours in medium free of the
drug. In general, the structure of these partially
resistant cells was unchanged. Some mitochondria
were swollen b u t were often found in close proximity to mitochondria of normal structure. Nucleoli
resembled those of the untreated sensitive line
(Fig. 7). Large nuclear inclusions, not seen in control cells, were present in a few cells. Phagocytosis
was more common, undoubtedly due to the increased number of dead cells in the cultures.
When these partially resistant cells were fixed
while still in media containing actinomycin D,
nucleolar alterations similar to those seen in sensitive cells were invariably present. Nucleoli appeared as small, round bodies with structureless
interiors, the majority of which were in the process
of division or budding. Nucleolar fragmentation
led to the appearance of many small round
nucleoli.
HeLa-R.--There were no apparent morphologic differences between resistant cells growing in
normal medium or those growing in medium con-
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JOURNEY
AND
GOLDSTEIN--HeLa Cells and Actinomycin D
raining 0.1 gg/ml of the drug. In general, resistant
cells were smaller, with less cytoplasm than sensitive HeLa cells. Occasionally, cells differing from
the typical epithelial type were found; these variants were long, spindle-shaped cells and were
usually isolated from other cells. There was no evidence of vesiculation of cell surfaces as seen in
sensitive cells. The most striking difference in
resistant cells was the appearance of the nucleolus.
Nucleoli of resistant cells were large and dense as
compared with the small, hyaline type found in
drug-treated sensitive cells (Fig. 8).
M a n y nuclei were lobulated and infolded and
in some instances contained membrane-bound
inclusions. Cytoplasmic organelles were similar to
those of control cells, although vacuolization was
common. Phagocytic vacuoles were frequently
present, either as large, empty vacuoles or numerous small secondary masses scattered through the
cytoplasm.
DISCUSSION
The principal cytotoxic effects in sensitive
HeLa cells treated with actinomycin D were: (a)
nucleolar alterations, and (b) blebbing and pinching-off of cytoplasm from the cell surface. Nucleolar changes were observed consistently in sensitive
and partially resistant HeLa cells in the presence
of very low concentrations (0.1 to 0.001 #g/ml)
and appear to be a specific effect of actinomycin
D. Fragmentation or budding of nucleoli and conversion to the lightly stained, hyaline type were
invariably present in drug-treated cells. These
observations confirmed and extended our previous
light microscope findings with histological and
histochemical staining procedures.
In general, nucleolar morphology is labile and
responsive to variations in the metabolic activity
of cells. Nucleoli are hypertrophied in embryonic
and other cells displaying active synthesis and reduced in quiescent cells. Structural changes can
also be produced by a wide variety of physical and
chemical agents (~6). For example, Hughes (17)
noted that several purine derivatives caused fragmentation of nucleoli in chick tissue cultures,
which reconstituted when cells were washed free of
drug. In this regard, most sensitive HeLa cells
quickly recovered when rinsed free of highly
cytotoxic concentrations of actinomycin D.
Suggestive evidence for the mechanism underlying altered staining affinity of nucleoli in drugtreated cells was obtained by chemical assays for
RNA, DNA, and protein. Preliminary experiments
revealed that both R N A and protein, the primary
constitutents of nucleoli, were depleted in sensitive cells, whereas D N A content was unchanged
(15).
931
Blebbing of the cell membranes suggested that
actinomycin D may act primarily at the cell surface. I t should be noted, however, that these
drastic surface changes were observed only occasionally. Although treatment with antibody
produced vesiculation of cell membranes in ascites
tumor cells (14) and HeLa cells, 1 these vesicles
were essentially devoid of cytoplasmic contents.
There is no available information on the site of
action of actinomycin D. To examine this problem, experiments with labeled actinomycin D are
planned to study its permeability in HeLa cells
and possible sites of intracellular accumulation.
Additional morphological features not previously described in HeLa strains were hyaline or less
osmiophilic regions and fibrous areas in the cytoplasm of some cells. These areas were not due to
actinomycin D, since they were found in control as
well as treated cell lines.
Edwards and Fogh (8) described structural
alterations in normal human amnion cultures produced by long exposure to trypsin. Among these
alterations were areas of fine filaments and hyaline
cytoplasmic regions, although no differentiation
was made between these two dissimilar entities.
Although brief exposure to trypsin was used
routinely in subculturing our HeLa lines, hyaline
regions were not a consistent finding. Recent evidence (~3) indicates that these hyaline regions
probably represent areas of glycogen deposition.
The presence of concentrically arranged fibrillar
areas has been noted in leukemic blast cells (3, 13),
solid (~, 9), and ascites tumor cells (1, 10). Similar
fibrils were found in our HeLa lines. These fibrous
components were more prevalent in resistant
HeLa cells, but their origin o r significance is uncertain.
The most remarkable cytological feature of
fully resistant HeLa cells was the persistence of
prominent, intensely osmiophilic nucleoli with no
evidence of fragmentation. There are several possible mechanisms by which cells might become resistant to actinomycin D: (a) cell surface changes
prohibit entry of the drug; (b) the drug is bound or
detoxified within the cell; and (c) induction of a
hydrolytic enzyme to inactivate the drug. In this
regard Katz and Pienta (18) have presented evidence which indicated that an enzyme system is
involved in the decomposition of actinomycin by
bacterial organisms. There is, at present, no evidence to indicate that higher organisms possess a
similar enzyme system.
The function of the nucleolus is incompletely
understood. Since actinomycin D appears to have
a specific effect on nucleolar morphology, it should
1Unpublished observations.
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Cancer Research
prove useful in exploring the role of this structure
in cell activities.
ACKNOWLEDGMENTS
The authors wish to acknowledge the assistance of Eva
Havas Pfendt and Charlotte Pollard.
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Fio. 1.--Living stock HeLa cell in normal medium. Note
the villi along the limiting boundary of the cell. Long filamentons mitochondria are scattered through the cytoplasm, and
dense lipide droplets are concentrated near the nucleus. The
nucleoli, which are out of focus, appear as dense ovoid bodies.
Positive phase contrast, )<1,600.
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I n Vitro Studies with HeLa Cell Lines Sensitive and Resistant to Actinomycin D. Ann. New York Acad. Sc., 89:
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FrG. 0.--Living stock HeLa cell incubated for 04 hours in
medium containing 0.1 gg/ml of actinomycin D. Note the
irregular ruffled contour of the cell boundary. There are
numerous blebs and cytoplasmic droplets (arrows) which have
broken away from the cell surface. The nucleoli have a mulberry-like configuration and appear smaller than those in
control cells. Positive phase contrast, X0-,000.
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FIG. 3.--Stock HeLa cell containing three dense nucleoli.
Numerous pleomorphic mitochondria (some appear vacuolated) and several tubules of tile endoplasmic rcticulum
(ER) are found in this section. In lower portion are profiles
of microvilli which project from surfaces of the neighboring
cells. X 1S,O00.
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~IG. ~.--Micrograph showing nucleolar changes in stock
HeLa cells after treatment with actinomycin D. The hyaline
interior arid dense peripheral granules are clearly evident,
and two buds have formed. X 13,000.
FIG 5.--Another variation of nucleolar fragmentation produced in stock HeLa cells by actinomycin 1). X 16,000.
FIG. 6.--Stock HeLa cell after exposure to 1 #g/ml actinomycin D. Cytoplasmic blebs in various stages of formation
and release are seen. Mitochondria are vacuolated, and nucleoplasm appears disorganized. Dense granular accumulations
in nucleus (N) m a y be either chromatin or remI~ants of disintegrated nucleoli. X7,000.
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FIG. 7.--Micrograph of partially resistant HeLa cells fixed
after ~4 hours in medium free of actinomycin D. Nucleolus
is densely stained; two nuclear indentations (ID) are indicated.
Several "lysosomal" granules (LY) and the Golgi complex
(G) are present. A hyaline area (HA) occupies a portion of the
cytoplasm in each cell. >(13,000.
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FIG. 8.--Micrograph of resistant HeLa cells, i.e., ceils that
grow and are maintained in media containing 0.1 ~g/ml
actinomycin D. Nucleoli of resistant cells appear dense and
intensely osmiophilic. X7,000.
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Electron Microscope Studies on HeLa Cell Lines Sensitive
and Resistant to Actinomycin D
L. J. Journey and Milton N. Goldstein
Cancer Res 1961;21:929.
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