[CANCER RESEARCH 38, 2290-2294.
0008-5472/78/0038-0000$02.00
August
1978]
Effects of Heat on the Centrosomes of Chinese Hamster Ovary Cells1
Marny D. Barrau, Gary R. Blackburn,
and William C. Dewey
Department of Radiology and Radiation Biology, Colorado State University, Fort Collins, Colorado 80523
ABSTRACT
Chinese hamster ovary cells were heated either at 45.5°
for 15 min or at 42°for 1 hr and then were either fixed
immediately or allowed to recover at 37°for intervals of up
to 12 hr. In addition control cells and cells heated at 45.5°
for 15 min were immediately subjected to a cell fractionation procedure that yielded partially purified centrosome
preparations. In 100% of the cells fixed and examined
immediately after heating, the centrosomes were dam
aged. The osmiophilic cloud increased in density and
became aggregated. The majority of the pericentriolar
particles or virus-like particles disappeared, and in some
cases the tubules of the wall of the centriole appeared
disrupted. These changes were also noted in the much
more abundant population of centrosomes in the partially
purified cell fraction. Furthermore, in those cells heated
at 45.5°for 15 min, no recovery of the centrosomes or
return of virus-like particles occurred even after incuba
tion at 37°for 12 hr.
INTRODUCTION
The increasing use of hyperthermia in cancer therapy has
generated interest in the effects of heat on cells in vivo and
in vitro (4). Heat treatment of cells in culture results in
mitotic delay. For example CHO cells heated for 6 min at
45.5° in G,, S, or G.¿
are delayed about 12 hr in reaching
mitosis, and this delay increases with an increase in either
the temperature or duration of heating (5, 30). CHO2 cells in
late prophase and metaphase are delayed from completing
division for the duration of the heating (5 to 7 min at 45.5°)but
recover as the cells return to 37°(3). However, those cells
heated in metaphase at 45.5°for 6 min are delayed by about
15 hr in reaching the next mitosis (5, 30). Even when these
cells reach mitosis, cytokinesis and often karyokinesis are
abnormal, since 90% are tetraploid, and about 50% have
more than 1 nucleus during the next interphase (5, 30). In
addition late-S and G, cells heated continuously
at only
41.5° are delayed in G2 for about 4 hr and then traverse
through metaphase at about one-third the normal rate (21).
However, cells heated in anaphase are not delayed in
completing division (23). Finally, high temperatures
have
been reported both to inhibit the polymerization
of microtubules (1, 12) and to cause their disassembly
(12, 24),
which results in a corresponding
loss of birefringence
of
the mitotic spindle (13).
Because of this evidence that heat damages the mitotic
apparatus, the effects of hyperthermia on a major compo1This work was supported by Grant CA 18334 awarded by the National
Cancer Institute, Department of Health, Education, and Welfare.
«The abbreviations used are: CHO, Chinese hamster ovary; MTOC,
microtubule-orgamzing center; VLP, virus-like particles.
Received August 26, 1977: accepted May 3,1978.
2290
nent of the mitotic apparatus, the centrosome, should be
worthy of investigation.
In CHO cells the centrosome
is
composed of a pair of perpendicularly
oriented centrioles
and an osmiophilic cloud or halo (10). Dispersed through
out the cloud are numerous small particles that morpholog
ically resemble viruses (10, 31). This integrated structure
apparently serves as a MTOC and thus plays a key role in
the formation of the spindle fibers and the separation of the
sister chromatids
during mitosis (10, 25). However, the
precise manner in which the centrosomes direct the forma
tion of the mitotic apparatus has not been determined.
In
fact it has not been clearly demonstrated whether it is the
centrioles themselves, the pericentriolar
material, or both
that are essential to mitosis (6, 10, 17, 25). However, a
recent study (10) of microtubule
formation
in CHO cells
indicated that the pericentriolar
material by itself can serve
as a nucleating site and that very little microtubule forma
tion originates at the centrioles.
Therefore, to determine whether there is a possibility that
G2 delay resulting from hyperthermia
might result from
damage to the centrosome, we examined in this preliminary
study the ultrastructure
of the centrosomes of control and
heat-treated CHO cells. The cells were heated either at 45.5°
for 15 min or at 42.0°for 1 hr, and centrosomal structure
was examined in thin sections of either whole cells or
partially purified cell subfractions.
In all cases significant
disruption of centrosome morphology was noted.
MATERIALS
AND METHODS
Cell Culture. Asynchronous CHO cells were cultured in
suspension in Bélicospinner flasks in McCoy's Medium 5A
containing 10% calf and 5% fetal calf sera along with the
following antibiotics: neomycin sulfate (0.1 g/liter); potas
sium penicillin G (0.05 g/liter); and streptomycin
sulfate
(0.05 g/liter).
Ultrastructural Studies on Whole Cells. The cells main
tained at pH 7.2 were heated either at 45.5°for 15 min or at
42.0°for 1 hr by immersing the spinner flasks containing 50
ml of medium in Precision Scientific water baths (accuracy,
±0.03°).Four min were required for the medium to reach
45.5°(half-time 0.85 min). Control flasks were placed in 37°
water baths for the same time interval. Two ml of medium
were removed immediately after heating and at intervals
after the spinner flasks were returned to 37°.
Centrosome Purification. The method for the purification
of centrosomes from CHO cells has been reported else
where (2).
Ultrastructural
Studies on Partially Purified Centro
somes. Because of the much larger number of cells re
quired for these experiments,
the heating protocol was
modified as follows. Approximately
10°cells in 1.8 liters of
growth
medium
were harvested
by centrifugation
CANCER
RESEARCH
at 500
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Heat and Centrosomes
rpm (78 x g) ¡na Mistral 6L centrifuge equipped with a J43
rotor. The cell pellets were then either resuspended in 25
ml of centrosome isolation buffer (controls) or in 1.8 liters
of growth medium preequi librai ed to 45.5°(heated cells).
After 15 min the heated cells were placed on ice, and then
centrifuged as described above. The pellets of the heated
cells were resuspended ¡n25 ml of centrosome isolation
buffer and, together with the controls, were submitted to
the centrosome purification procedure.
Electron Microscopy. The cells or purified centrosomes
were centrifuged
in the cold, fixed in phosphate-buffered
1%OsO4, rapidly dehydrated in graded ethanol, and embed
ded in Epon 812 (9). Thin sections were stained with uranyl
acetate and lead citrate (18) and were examined with a
Philips Model 200 electron microscope.
RESULTS
Ultrastructural Studies of Centrosomes in Sections of
Whole Cells. Fig. 1 illustrates centrosomes from control
cells. The centrioles are surrounded
by a lightly stained
fibrous cloud. Embedded in the cloud are 60-nm VLP that
typically have a dense core of approximately
35 nm and a
lighter peripheral halo. The centrioles are most frequently
seen ¡noblique sections, but their tubular nature is still
evident.
t y»,
v»
-
BB
Immediately
after heating
the cells at 45.5° for 15 min
(Fig. 2) the appearance of 100% of the centrosomes was
altered (25 centrosomes
were studied). The osmiophilic
halo had aggregated and was more electron dense than in
controls. A few VLP were seen but most had disappeared.
Similar changes were observed in 100% of the centrosomes
in cells heated at 42.0°for 1 hr.
When the cells heated at 45.5°for 15 min were incubated
from 15 min to 12 hr at 37°no further changes were noted
in 25 abnormal centrosomes examined (Figs. 3 and 4); i.e.,
the osmiophilic cloud was still dense and aggregated, and
only a few VLP were discernible. Apparently, even 12 hr of
recovery at 37°is inadequate to allow repair of the damaged
centrosome structure.
With respect to alterations in the structure of the cen
trioles themselves, the density of the pericentriolar
material
in the heated cells made it difficult to detect damage to the
tubules of the centriolar
wall. In some cross-sections,
however, the tubules did appear to be distorted (Fig. 3)
whereas in others, such as that shown in Fig. 4, the cen
trioles seemed to have a normal tubular arrangement.
Utrastructural Studies of Centrosomes in Partially Puri
fied Cell Fractions. The alterations in centrosome structure
observed in sections of heated whole cells were confirmed
by examination
of partially purified centrosomes derived
from cells heated at 45.5°for 15 min. However, in the latter
A •¿
i
S&c:«
Fig. 1. Centrosomes (C) from control cells. The centrioles are surrounded by electron-dense osmiophilic clouds (H). VLP (large arrows) with a dense core
and lighter periphery are embedded in the osmiophilic clouds. Some of the tubules of the centriolo can be distinguished (small arrows), x 74,000.
Fig. 2. The centrosome of a cell fixed immediately after heating for 15 min at 45.5°.The osmiophilic cloud (H) is aggregated and dense, especially in
juxtaposition with the centriolo (C). Only occasional VLP (arrow) are discernible, x 74,000
AUGUST
1978
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2291
M. D. Barraci et al.
case literally hundreds of centrosomes could be scanned in
a short time due to the enrichment obtained by the purifi
cation process. Thus, the damage observed in the relatively
few centrosomes examined in the "whole-cell" portion of
this study is almost certainly a lesion common to all cells
treated as described.
In addition to the damage evident in the centrosomes
observed in situ, i.e., aggregation of pericentriolar material
and loss or partial disruption of VLP, the heated centro
somes in the purified preparation also showed more clearly
significant damage to the characteristic 9-fold triplet orga
nization of the centriole itself. The example in Fig. 6
illustrates a characteristic common to many of the heattreated centrioles observed, i.e., the presence in the crosssection of a relatively amorphous pair of concentric circles
replacing the well-ordered triplet tubule vanes (illustrated
in Fig. 5 fora control).
DISCUSSION
delay must be correlated with the time intervals required for
recovery from pericentriolar damage.
The remaining elements of CHO centrosomal structure,
the small particles embedded in the pericentriolar material,
have been compared to type A or precursor type C RNA
oncornaviruses (10, 31) and thus may be associated with
the transformation of CHO cells. Furthermore, the hypoth
esis has been presented that the association of the VLP
with the pericentriolar material serves to distribute auto
matically the oncornaviruses to daughter cells during divi
sion (31). Since the evidence is quite convincing that the
VLP are not essential to the MTOC function of the pericen
triolar material (10) [also 12 cell lines studied did not have
any VLP (31)], attributing an alternative viral function to
these particles is reasonable.
The suggestion has been made that many cells, including
CHO, contain a "quiescent" oncornavirus that can be
stimulated to transform cells or produce tumors (26, 27).
Moreover, a small level of RNA-directed DMA polymerase
activity has been detected in CHO cells (26). By the same
token Wheatley (31) has suggested that the VLP associated
with centrioles might be an inducible oncornavirus. If this
is true the disappearance of the VLP after hyperthermic
treatment could lead to an alteration in the transformed
state of the CHO cells. Obviously, many experiments are
needed to determine whether the VLP are simply displaced
from the pericentriolar material or are destroyed by heat
treatment, as has been reported in the thermal inactivation
of other viruses (7, 8, 28). Once the fate of the VLP after
hyperthermic disruption has been established, their pres
ence or absence may be correlated with the transformed
state of the CHO cells in question.
This study has shown that hyperthermia induces signifi
cant changes in centrosome structure. Do these changes
correlate with specific damage to cellular function; e.g., do
they contribute to cell lethality or mitotic delay? Moreover,
can the apparent damage to the VLP associated with the
pericentriolar material be of consequence in inducing these
pathological states? On the other hand, if damage to the
VLP does not affect normal cell processes, what is the
metabolic function (if any) of these particles?
With regard to the observed changes in the centrioles
themselves and the amorphous pericentriolar cloud the
latter material apparently serves as a MTOC (10, 16, 17, 29)
and is thought to be important for formation of the mitotic
spindle (11, 20, 22) and maintenance of cell shape during in
terphase (15). In fact the pericentriolar material appears to be
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2293
•¿(D
Fig. 3. Cross-section of a centriole from a cell allowed to recover for 15 min at 37°after heating at 45.5°for 15 min. The tubules (arrow) of the wall of the
centriolo appear abnormal (compare with Fig. 5). x 74,000.
Fig. 4. Cross-section of a centriole from a cell allowed to recover for 12 hr at 37°after heating at 45.5°for 15 min. The centriole appears to have the typical
arrangement of tubules (9 sets of 3; compare with Fig. 5). However, there is a large amount of densely aggregated material (H) surrounding the centriole.
x 74,000.
Fig. 5. Cross-section of a centriole isolated from control cells The 9-fold triplet symmetry is evident, x 60,000.
Fig. 6. Cross-section of a centriole isolated from cells heated at 45.5°for 15 min. Disruption of the 9-fold triplet symmetry is evident. Also, the amorphous
contaminant seems to be aggregated, x 60,000.
2294
CANCER
RESEARCH
VOL. 38
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1978 American Association for Cancer Research.
Effects of Heat on the Centrosomes of Chinese Hamster Ovary
Cells
Marny D. Barrau, Gary R. Blackburn and William C. Dewey
Cancer Res 1978;38:2290-2294.
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