[CANCER
RESEARCH
28, 521-534, March 1968]
Ultrastructure of Tumor Cells during Mitosis1-2
Jeffrey P. Chang and Charles W. Gibley, Jr.8
Department
oj Pathology,
The University of Texas M. D. Anderson Hospital and Tumor Institute
SUMMARY
The fine structure of the mitotic process of solid hepatoma
cells of rat has been described. The replication and migration
of centrioles, appearance and disappearance of spindle tubules
and kinetochores, and movement and replication of chromo
somes resemble those reported in other cell systems.
It has been observed that the nucleoli persist throughout all
stages of cell division. The Golgi apparatus retains its discrete
entity of laminar and vesicular components during the entire
mitotic cycle. Remnants of the nuclear envelopes have been
observed in all phases, even in telophase, after the reconstruc
tion of the nuclear membrane of the daughter cells is com
pleted. Penetration of rough endoplasmic reticula into the
mitotic apparatus may appear in metaphase and anaphase. In
telophase, cytokinesis is initiated by symmetrical invagination
of the plasma membrane and, at certain stages, mitochondria
may move freely in the region of the intercellular bridge which
connects the daughter cells.
The mitotic activities of these tumor cells represent the be
havior of the cellular organelles in natural and unaltered nu
tritional environment.
INTRODUCTION
The fine structure of mitosis and meiosis has been studied in
a wide variety of plant and animal cells, including onion root
tip (33), spermatocytes (16, 24, 31), meristem of wheat (32),
developing embryos (1, 18, 19, 20), protozoans (15, 38-40),
and several types of tissue culture cells (6-8, 17, 26, 36). Re
ports of investigations of mammalian cell division have been
limited in number (10, 14, 30). Electron microscopic observa
tions of the mitotic cycle in tumor cells have been scanty and
offer only fragmentar}7 information. Buck (9) described the
lamellae in the spindle of mitotic cells of Walker tumor. Yasuzumi (47) studied the nuclear membrane in prophase and
telophase of Yoshida sarcoma cells. Selby (42) published elec
tron micrographs of low magnification (X 3300-4200) of mi
totic figures of cultured Ehrlich ascites cells. Due to lack of
adequate technic at that time, important cellular organelles
1 This research has been supported by USPHS Grant, CA05312, from National Cancer Institute.
2 Reported at the Annual Meeting of the Society for Cell Biol
ogy, November, 1966, Houston, Texas.
3 Present address : The Pennsylvania College of Podiatric Medi
cine, Pine at Eighth Street, Philadelphia, Pennsylvania 19107.
Received July 5, 1967; accepted November 13, 1967.
at Houston,
Houston,
Texas 77025
such as mitochondria, Golgi bodies, nucleolus, and centrioles
could not be identified in these micrographs. It appears that
systematic study of fine structure of mitotic activity of solid
tumor cells in their natural environment is lacking. This re
search was thus undertaken to fill the need. Data from this
investigation would be of special value and interest when com
pared with those from cultured cells.
MATERIALS AND METHODS
Induction of primary hepatoma by feeding rats the carcin
ogen, 3'-methyl-4-dimethylaminoazobenzene,
and the establish
ment of transplantable hepatomas from this tumor have been
described elsewhere (12). In the present study, tissues of the
transplantable tumor were fixed in 3% glutaraldehydc (41) or
in a glutaraldehyde-acrolein mixture at 4°Cor at 25°C,and
postfixed in 1% osmium tetroxide. All fixatives were buffered
with Millonig's (29) phosphate buffer at pH 7.4. After fixation
for 90 minutes, the tissues were washed in tap water contain
ing a trace of calcium chloride and were dehydrated rapidly
with ethyl alcohol by the drop-wise replacement method. Re
placement of the ethyl alcohol by propylene oxide preceded
embedding in Epon 812 (28). Thick sections (0.5-1 ¡i)stained
with méthylène
blue and azure II (35) were used to locate
mitotic cells and to identify the tumor type as well as the
exact mitotic stage at the level of the light microscope. The
tumor cells studied were carcinomas. Thin silver or gray sec
tions were stained with uranyl acetate (45) and lead citrate
(34) and examined with an Hitachi HU-11A electron micro
scope.
OBSERVATIONS
Interphase
The fine structure of the interphase cell in the primary tu
mor and in the transplants has been described in a previous
paper (11). The nuclear pattern and the extent of the cyto
plasm vary considerably; general features, however, are illus
trated in Fig. 1. The nuclei are round or irregular in shape and
may occupy a large portion of the cell. The chromatin and
the nuclear envelope are closely associated. Nucleoli are large,
irregular in shape, and are located centrally or peripherally.
They are composed of the usual granular and fibrillar elements
(the pars granulosa and the pars fibrosa). The cytoplasm,
which is of moderate amount, contains scattered mitochondria,
well-developed Golgi areas, and a heavy population of free
ribosomes. Microbodies are rarely observed, yet lysosomes are
abundant, usually near the Golgi region. Elements of rough
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521
Jeffrey P. Chang and Charles W. Gibley, Jr.
endoplasmic reticulum are scattered through the cytoplasm;
usually, they are not stacked in parallel fashion, as are those
in normal hepatocytes. Glycogen particles have not been posi
tively identified in any sections thus far examined. Centrioles
are often found in the tumor cells in the vicinity of the Golgi
complex. Each centriole is composed of nine radiating groups
of tubules in triplets, arranged to form a cylinder (Fig. 1
insert) as previously described (4, 36, 44). At interphase, four
centrioles are located in a small hilus in one side of the nucleus.
Prophase
Condensations of chromatin into discrete chromosomes along
the nuclear membrane characterize prophase (Figs. 2, 3). The
association of the nuclear envelope and the chromatin becomes
less pronounced as prophase is approached. On some sections,
the kinetochore may be visible (Fig. 2). As cell division pro
ceeds, the nuclear membrane gradually breaks into pieces.
These remnants have also been observed in other stages of
mitosis. Often the two smooth sides of remnants come in con
tact with each other (Fig. 3). This feature corresponds to the
duplication of the nuclear envelope in rat lymphocytes (30)
and the "stacking" of the envelope reported by others (2).
In early prophase, migration of the centrioles toward the
opposite poles takes place in the usual manner. On rare oc
casions, more than two centrioles have been seen at one polar
region in a prometaphase (Fig. 4). This phenomenon resembles
the observation on colcemid-treated Chinese hamster cells (8).
Its cause is not clearly understood.
The behavior of the nucleolus in prophase is similar to that
of the Chinese hamster cells (6). In essence, the organdÃ-es
gradually undergo morphologic transformation and lose their
fibril element, and the granular elements become more uni
formly and distantly dispersed. Eventually, they break into
smaller masses; usually these masses become attached to con
densed chromosomes, although they have been found free in the
cytoplasm. This condition persists throughout metaphase and
anaphase.
The Golgi systems are well defined in prophase. They exhibit
both laminar and vesicular components (Fig. 3). Also, they
tend to remain in the polar regions (Fig. 3), although occasion
ally they are observed in other locations.
The spindle tubules are not detected until late prophase. The
centrioles are then firmly situated in the polar region, while
the chromosomes have moved toward the equatorial plate.
strates one endoplasmic reticulum in contact with the tubule;
the latter, though poorly sectioned, is identifiable for a short
distance and is attached to a kinetochore. The proliferation of
endoplasmic reticulum into mitotic apparatus of root-tip cells
of onion has been reported (33). Smooth-surfaced membrane,
resembling the cisternae of the endoplasmic reticulum, has also
been observed in metaphase of HeLa cells (36).
Remnants of nuclear envelope are scattered in the cytoplasm.
A few mitochondria are apparent in the mitotic apparatus,
at times near the chromosomes (Figs. 6, 7). The Golgi appa
ratus retain their identity in metaphase and are usually local
ized in the two polar regions (Fig. 5). Both the laminar and
vesicular forms are often detected in different planes of section.
Finally, the nucleolar material always maintains its identity
as small masses attached to the chromosomes (Figs. 5-7). This
phenomenon is clearly demonstrated in Fig. 7, which shows a
polar view of a section cut through the equatorial region. The
morphologic characteristics of the nucleolus are similar to those
previously described (6).
Anaphase
The ultrastructural features of early anaphase closely resem
ble those of late metaphase. In ultrathin sections, separation
of these phases into two distinct stages is extremely difficult
(3). Figures 9 and 10 show the relationship between the cen
triole and the chromosomes, the arrangements of spindle tu
bules and proliferating endoplasmic retÃ-cula,and mitochondria
near the anaphase chromosome. The spindle tubule may be
seen attached to the kinetochore. In Fig. 10 the Golgi appa
ratus is clearly apparent in the polar region. As cell division
progresses, the chromosomes move toward the centrioles, and
the nucleolar masses become more prominent and more elec
tron opaque. A well-organized Golgi system and occasional
stacked nuclear envelopes are also observed. When the two
masses of chromosomes separate still farther, the reconstruction
of nuclear membrane becomes obvious. At times, a number of
membrane-bound electron-opaque droplets are discovered scat
tered through the cytoplasm (Fig. 11). These bodies, presum
ably lipid in nature, correspond to the membrane-bound osmiophilic bodies described by Robbins and Gonatas (36) ; as a
rule, however, they do not form clusters in the tumor cells
and are rarely found in prophase. In many sections they be
come more numerous in anaphase.
Telophase
Metaphase
In this stage the chromosomes are aligned on the equatorial
plate and the spindle tubules form a cup-shaped umbrella
radiating from the poles where the centrioles are located. The
tubules, varying in number, may attach to or penetrate be
tween the chromosomes (Figs. 5, 6). During this stage seg
ments of proliferating endoplasmic reticulum may lie parallel
to the spindle tubules. The two organelles can be identified
easily: the former have a varied diameter, a somewhat un
dulating profile, and attached ribosomes, whereas the latter are
always uniform in width, have a rigid profile, and lack ribo
somes. Figure 8 shows a number of the endoplasmic retÃ-cula
radiating from the pole toward the chromosomes. It demon
522
The typical telophase is illustrated in Figs. 12 and 13. The
chromosomes condense into highly electron-opaque masses at
each pole. The newly formed nuclear membrane with nuclear
pores gradually surrounds the chromosome. The nucleolus may
be seen embedded in the mass of chromosomes; usually it is
located at the periphery of the nucleus. At the late stage, the
granular and fibrillar components of the nucleolus may be ob
served (Fig. 16). The Golgi apparatus with both laminar and
vesicular elements generally are well developed; they are lo
cated between the daughter nuclei and the equatorial plate
(Fig. 13).
Cytokinesis takes place by symmetrical invagination of
plasma membrane in the late telophase of these tumor cells
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Tumor Cell Mitosis
(Fig. 12). The cytoplasm forms an intercellular bridge between
the two daughter cells just before they separate (Figs. 13-15).
As a rule, numerous spindle tubules converge into a dense
zone, the midbody, as they pass through the bridge (Fig. 15).
Numerous electron-dense granules aggregate outside these tu
bules. Occasionally, mitochondria may be found in the region
of the midbody where there are few spindle tubules (Fig. 14).
No information has been obtained to indicate how the midbody is eliminated when the daughter cells finally separate.
DISCUSSION
The authors have encountered many difficulties in conduct
ing this research because of the scarcity of suitable mitotic
figures in a given section and the variable incidence of differ
ent phases of cell division. Our efforts were worthwhile, since
data obtained from the study revealed the cellular activities
of tumor cells in a natural and unaltered nutritional environ
ment. Thus far, the results indicate that the replication and
migration of centrioles, the formation and dissolution of spindle
fibers and kinetochores, and the movement and replication of
chromosomes resemble those in other cell systems, although
inconsistent variations are observed. Certain organelles in the
tumor cells, however, exhibit a characteristic behavior during
mitosis.
Hay (21) stated that persistent nucleoli are common in mi
totic cells of plants, though normally the nucleoli do not persist
during mitosis in animal cells. Heneen and Nichols (22) be
lieved that the nucleoli persisted in mitotic cells but were not
included in daughter nuclei of mammalian cells; they either
degenerated in the cytoplasm or were eliminated from the
cell. Lafontaine and Choninard (27) reported that in Vicia
¡aba the nucleolus dissolves completely during late prophase and becomes indistinguishable from other nuclear and
cytoplasmic material; further, the fibrillar and granular forms
of the nucleoli reappear along the arms of anaphase chromo
somes. Similar observations in grasshopper were published by
Stevens (43). In autoradiographic, cytochemical, fluorescent,
electron microscopic, and ribonuclease digestion examinations
of a number of mammalian cells, Hsu et al. (23) observed that
persistent nucleoli, having the same characteristics as the
nucleolus in interphase, could be identified in all the stages of
mitosis. In a detailed ultrastructural study of Chinese hamster
cells, Brinkley (6) found that in prophase, nucleoli disperse
into smaller masses. These masses appear as a loose, pre
dominantly fibrous structure with widely scattered granules.
This structure persists throughout mitosis; in telophase it
becomes included in the daughter nucleoli.
In the present study of the tumor cells, although nucleolar
particles were dispersed and discrete fibrillar areas were lost
during the mitotic cycle, nucleolar material was found during
each stage (Figs. 2, 5-7, 9, 16). Figure 16 demonstrates a nu
cleolar mass containing both granular and fibrillar material in
a separating daughter nucleus. This indicates that the persist
ent nucleolus enters the daughter cell. One cannot, however, dis
regard the possibility that the nucleolus is newly organized in
telophase. The functional significance of persistent nucleoli in
mammalian cells is not clear. It was thought (6) that they
serve either as a primer for new nucleolar material or as a
source of nucleolar RNA for the daughter cell during the for
mation of a new nucleolus.
The exact disposition of the nuclear envelope during mitosis
is still an open question. In the amoeba (39) the membranes
of the nuclear envelope are present in all stages but are not
continuous in metaphase. In rat thymocytes a complete ring
of nuclear envelope has been observed in late prophase and
early metaphase; even at anaphase large segments have been
recognizable (30). Although large segments of the nuclear en
velope are not visible in solid tumor cells, remnants of it are
clearly present in all phases of the mitotic cycle (Figs. 3, 5,
13). Buck (9) detected lamellar structures in the spindle area
of the mitotic cells of Walker 256 carcinosarcoma. He believed
that these structures were similar to the endoplasmic reticulum
and represented a newly forming nuclear membrane. Barer
et al. (2) also suggested that the new nuclear membrane might
be formed by a fusion of remnants of the endoplasmic reticu
lum. In human lymphocytes, the nuclear membrane is believed
to reform from a fusion of vesicles derived from the old nuclear
membrane (25). It is possible, therefore, that the new en
velopes are reassembled either from the remnants of the old
nuclear envelope or from endoplasmic reticulum, or from both.
The findings in the present study do not strongly support any
of the theories in particular, since both the stacked remnants
of the nuclear envelope (Figs. 3, 5) and the proliferating endo
plasmic reticulum into the spindle (Figs. 8, 9) were often ob
served. In late telophase, after the reconstruction of nuclear
membrane is completed, an abundant amount of the stacked
nuclear membrane is occasionally present in the cytoplasm
(Fig. 13). This stacked material will probably either degenerate
or transform into normal endoplasmic reticulum in interphase.
The fate and function of the Golgi apparatus in mitotic cells
is another enigma. In a review of the Golgi complex, Bourne
and Tewari (5) stated, "In mitosis the Golgi apparatus usu
ally breaks up into small particles or granules which are dis
tributed more or less evenly through the cytoplasm." Dalton
(13) also reported the absence of Golgi elements in several
tumor cells during mitosis. In HeLa cells, Robbins and Gonatas
(36, 37) presented histochemical and fine structural evidence
that the Golgi disappears during metaphase and appears rapidly
in telophase. Conversely, Dougherty (14) observed the per
sistence of the Golgi complex during mitosis of hepatic cells in
rats. Roth et al. (40) reported unmistakable evidence that
typical Golgi bodies appear in metaphase and anaphase in
both the spermatocytes of European corn borer and the giant
amoebae. In our study, the Golgi apparatus did not break into
small vesicular clusters, but retained its laminar and vesicular
components throughout different phases of the mitotic cycle
(Figs. 3, 5, 10, 13).
The separation of the daughter cells is evidently initiated by
a symmetrical invagination of the cytoplasmic membranes. No
visible signs of specific morphologic changes of cellular or
ganelles in the cytoplasm are detected in this process. The
phenomenon described by Whaley et al. (46), that the Golgi
apparatus is clearly involved in the formation of cell plate
during cytokinesis, does not apply to the hepatoma cells. Evi
dently, a difference in species explains the differences in be-
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523
Jeffrey P. Chang and Charles W. Gibley, Jr.
havior of these organelles. At the end of telophase, the spindle
tubules forming the midbody are condensed in the region of
the intercellular bridge (Fig. 15). The structure of the midbody is similar to that of the HeLa cells (36). However, the
disposition of the midbody in the hepatoma cells is not clear.
The pincer-like invagination of the plasma membrane (36) or
the enclosure by the furrow membrane and its subsequent
abandonment (1) have not been observed in our system. On
occasion, mitochondria appear within the intercellular bridge
(Fig. 14) where no midbody is formed. This could represent
a different type of cytokinesis, or could be caused by section
ing at a different plane or at a different stage of the develop
ment of the midbody.
It may be concluded that rat hepatoma cells possess some
fundamental features of mitotic division similar to those of
other species. Data obtained so far indicate the persistence of
nucleoli and Golgi systems throughout the mitotic cycle. In
addition, proliferation of endoplasmic reticulum into the mi
totic apparatus, dissolution and reconstruction of nuclear mem
brane, and initiation of cytokinesis and formation of midbody
in telophase are observed in the hepatoma cells. It is not known
whether manifestations of these characteristics are controlled
by genetic or nutritional factors. Further elucidation of these
problems awaits the completion of other studies now in
progress.
ACKNOWLEDGMENTS
The authors are indebted to Dr. Charles W. Philpott, Rice Uni
versity, and Dr. Bill Brinkley for suggestions and criticisms, and
to Mr. Clemmon Woodard for his technical assistance.
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1968
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525
Jeffrey P. Chang and Charles W. Gibley, Jr.
Figs. 1-16. Tissues were fixed in glutaraldehyde-acrolein
mixture (unless stated otherwise) and postfixed in osmium tetroxide. All
sections were stained with uranyl acetate and lead citrate.
Fig. 1. Interphase cell. Showing irregularly shaped nucleus (N) and nucleolus (n) with fibrillar (/) and granular (g) components,
elements of the endoplasmic reticulum, and a number of mitochondria. Note the well developed Golgi (G). X 37,000. Insertion is a
cross section through a centriole. X 1,255,600.
Fig. 2. Prophase. Showing condensation of chromatin into discrete chromosomes (Ch) along the nuclear membrane (Nm). Note the
kinetochore (K) and the homogeneous nucleolus (n). X 18,270.
Fig. 3. Advanced prophase. Showing the breakage of the nuclear membrane and pieces of its remnants (arrows) having their smooth
sides joining. Laminar and vesicular elements of the Golgi (G) apparatus are shown. A similar mass of the Golgi (not shown) is found
on the opposite side of the nucleus. Note the lysosome granules in the Golgi area. X 21,420.
Fig. 4. Prometaphase. Showing three centrioles (arrows) at the polar region and masses of chromatin material. X 32,900.
Fig. 5. Metaphase. Tissue fixed in glutaraldehyde showing the complete mitotic apparatus. Laminar and vesicular forms of the Golgi
complex (G) are located at the poles. Chromosomes (Ch) are aligned on the equatorial plate. Note the nucleolar mass (n) the rem
nants of the nuclear membrane (Nm), and the dense granules near the Golgi. X 24,380.
Fig. 6. Metaphase. Showing the chromosomes (Ch) aligned on the equatorial plate, the nucleolus (n), kinetochore (K), etc. x 24,480.
Fig. 7. Polar view of metaphase. Showing the nucleolar (n) material, endoplasmic reticulum (ER), and mitochondria in the equa
torial region. X 16,720.
Fig. 8. Late metaphase. Showing endoplasmic reticulum (ER) in the area of the mitotic apparatus. A spindle tubule (SO appears
with an ER and attached to a kinetochore (K). X 44,880.
Fig. 9. Ana-phase. Showing chromosomes moving to the poles. Radiating from the centriole (C) are spindle tubules (St) and
endoplasmic reticulum (ER). X 35,520.
Fig. 10. Anaphase. Showing the relationship between the centriole (C), the chromosomes, and the spindle tubules. The Golgi appara
tus (G) is evident in the polar region. X 42,300.
Fig. 11. Late anaphase. Showing chromosomes (Ch) situated at their respective poles. A number of membrane-bound
electronopaque droplets (arrows) are scattered through the cytoplasm. X 20,060.
Fig. 12. Early telophase. Showing at each pole, the condensed chromosomes (Ch) with newly formed nuclear membrane (Nm).
Cytokinesis is evident (arroir). A symmetrical invagination (not shown) appears at the opposite side of this cell. X 19,470.
Fig. 13. Late telophase. Showing daughter nuclei with complete envelopes. Remnants of the excess nuclear membrane (Nm) remain
¡nthe cytoplasm. Note the midbody (Mb), the intercellular bridge (Ib), and the Golgi (G). X 15,120.
Fig. 14. Late telophase. Showing mitochondria (M) moving freely in the intercellular bridge where few spindle tubules (arrows) exist.
X 33,120.
Fig. 15. Higher magnification of a midbody of another cell showing numerous microtubules. X 30,100.
Fig. 16. Late telophase. Showing the centriole (C), the newly formed nuclear membrane, and the nucleolus (n) which contains ma
terial suggesting fibrillar (/) and granular (g) components. X 29,240.
526
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VOL. 28
Ultrastructure of Tumor Cells during Mitosis
Jeffrey P. Chang and Charles W. Gibley, Jr.
Cancer Res 1968;28:521-534.
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