[CANCER RESEARCH
46, 324-327, January 1986]
Hyperthermia-induced
Cell Death, Thermotolerance, and Heat Shock Proteins
in Normal, Respiration-deficient,
Cells1
and Glycolysis-deficient Chinese Hamster
Jacques Landry,2 StéphanieSamson,3 and Pierre Chrétien
Department ot Medicine, Laval University, and Laval University Cancer Research Center, Hôtel-Dieu de Québec,Québec,Canada G1R 2J6
ABSTRACT
Hyperthermia-induced
cell inactivation,
thermo-tolérance de
velopment, and heat shock protein synthesis were characterized
in a Chinese hamster cell line and its two derivatives, respec
tively, deficient in glycolysis and respiration. No difference was
found in the intrinsic thermosensitivity of the cells or in their
ability to respond to heat by developing thermotolerance and
synthesizing the heat shock proteins. The results indicate that
the direct thermal inactivation of either respiration or glycolysis
is not an obligatory rate-limiting event in hyperthermic cell killing
and that thermotolerance and heat shock proteins are not trig
gered exclusively as a consequence of damages induced directly
by heat in energy metabolism nor do they result specifically in
an increased thermostability of respiration or glycolysis. It is
concluded that if energy metabolism is involved somehow in the
induction by heat of cell death, thermotolerance, and heat shock
protein synthesis, it is due to a heat-induced alteration that
indirectly causes changes in the cellular energy status rather
than to a direct interaction of heat with energy production.
INTRODUCTION
In virtually all cells and organisms mild heat treatments induce
an enhanced synthesis of a family of polypeptides called HSP4
and the development of thermotolerance, a transient cellular
state of extremely high resistance to heat shock (1-5). Despite
extensive studies on these phenomena the nature of the initial
heat interaction or signal that triggers these specific cell re
sponses remains largely unknown (6, 7).
Beside heat, many chemicals and experimental conditions that
perturb energy metabolism also yield induction of HSP and
thermotolerance (2, 8, 9). Glucose or oxygen deprivation, for
example, induce under appropriate conditions both HSP synthe
sis and thermotolerance (10-12). Glycolysis and respiration are
rapidly inhibited in severly heated cells (13-15), and it was
suggested that a lethal lack of energy may be the primary cause
for hyperthermic cell killing (16). Furthermore, thermotolerance
was shown to result in an increased capacity of the cells to
survive prolonged deprivation in energy substrates (16) and to
maintain their ATP level upon subsequent heat treatments (17).
Based on these and many other observations (reviewed in Refs.
MATERIALS AND METHODS
The cells used in this study were obtained from Dr. Jacques Pouysségur,Nice, France. The DS7 and GSK3 clones were derived from 023
cells, an anchorage independent and tumorigenic subclone of the
Chinese hamster lung fibroblast cell line CCL39 (ATCC). Details of their
selection and characterization can be found in previous reports from this
group (19, 20). Briefly, DS7 is deficient in aerobic glycolysis and glucose
transport as a consequence of a block in phosphoglucose isomerase
activity. It does not produce lactic acid, and it derives its energy strictly
from respiration. GSK3 is defective in oxidative metabolism, has a
reduced consumption of oxygen, and depends exclusively on glycolysis
for growth and survival. O23 is the parental cell line with normal energy
Received 5/14/85; revised 8/23/85; accepted 9/18/85.
1This work was supported by the Medical Research Council of Canada, grant
MA-7088.
2 Chercheur-boursier du Fonds de la Recherche en Santédu Québec.To whom
requests for reprints should be addressed, at Centre de Recherche, l'Hôtel-Dieu
de Québec,11, côte du Palais, Québec,Canada, G1R 2J6.
3 Recipient of a studentship from the Fonds de la Recherche en Santédu
Québec.
4 The abbreviation used is: HSP, heat shock protein(s).
CANCER
RESEARCH
6 and 7) it was repeatedly proposed that energy metabolism
could be the primary target of the heat action, the site from
which originates the triggering signal for HSP and thermotoler
ance induction and the key metabolic pathway protected in
thermotolerant cells.
If alterations induced by heat in energy metabolism are in fact
the primary events in the induction of HSP, thermotolerance, and
cell death, then a cell deficient in either respiration or glycolysis
should respond to a heat treatment differently than its normal
counterpart that can rely on both pathways for its energy supply.
Indeed, if glycolysis is a prime site of hyperthermic damage,
respiration-deficient cells, because they cannot count on respi
ration to recover treatments, should be more heat-sensitive than
are control cells. On the other hand, glycolysis-deficient cells
would be expected to be more heat-sensitive than are control
cells if mitochondria are very sensitive to elevated temperatures.
Furthermore, if thermotolerance and HSP are triggered specifi
cally by alterations in one of the energy-producing pathways it is
predictable that glycolysis- or respiration-deficient cells will show
a modified capacity to become thermotolerant and to synthesize
HSP.
In a previous study we found no difference in the heat response
of respiration-deficient chick fibroblasts as compared to control
cells in terms of thermosensitivity, development of thermotoler
ance, and induction of HSP (18). This suggested that inactivation
of respiration or shift from an aerobic to an anaerobic type of
metabolism was not essential in this system for signalling the
induction of thermotolerance and HSP and, furthermore, that if
energy metabolism is the primary site of the lethal action of
hyperthermia, inactivation of glycolysis might be the rate-limiting
step in thermal inhibition of energy production. Here we compare
the response to heat of 2 mutants deficient in respiration and
glycolysis, respectively, to that of their common parental Chinese
hamster cell line. The results reveal no major difference in the
heat responses of the glycolysis-deficient cells as compared to
the respiration-deficient or control cells.
VOL. 46 JANUARY
1986
324
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ENERGY METABOLISM AND CELL RESPONSE TO HEAT
metabolism. The cells were propagated at 37°Cin a 5% CO2 humidified
atmosphere in Dulbecco's modified Eagle's medium containing NaHCO3
(2.2 g/liter) supplemented with 10% fetal bovine serum, penicillin (50 ID/
ml), and streptomycin (50 ng/ml). In some experiments, the cells were
maintained for various periods of time in a "glucose-free" medium made
of high-GEM medium (Flow Laboratories), a Dulbecco's modified Eagle's
medium in which fructose (3.6 g/liter) is substituted for glucose, supple
mented with 10% dialyzed fetal bovine serum (Grand Island Biological
Co.). We estimated that the dyalized serum contained less than 5 mg/
liter of glucose so that the final concentration of glucose in the serum
supplemented "glucose-free" medium was less than 50 Mg/100 ml. All
u-*
• 2
other procedures used are as before (4, 5,18) and are described briefly
in the legends to illustrations. |;KS|Methionine (translation grade, 1000
« » t
If
12 14 16 1*
Ci/mmol) was purchased from New England Nuclear and used at a
concentration of 25 ¿iCi/mlin standard medium.
RESULTS AND DISCUSSION
In many cell systems it has been demonstrated that respiration
and glycolysis are tightly co-regulated to insure sufficient supply
of energy in cases where one pathway is functioning at low
regime (e.g., under specific substrate deprivation) and that a cell
can survive complete inhibition of any one of the two energy-
2
«
Tim« of
B
B
incubation
11
12
14
(hour.)
Chart 2. Relative survival of O23 (•),
DS7 (•),
and GSK3 (A) cells as a function
of time in an energy metabolism restrictive medium. Cells exponentially growing in
normal medium were transferred at time 0 to glucose-free fructose-containing
medium (A) or to normal medium containing 0.5 ng of oligomycin per ml (B). At the
time indicated, the cells were trypsinized and plated at an appropriate concentration
in normal medium for colony formation. The arrow in ßindicates a time-point at
which no colony was obtained (survival is lower than 5 x 10"4).
producing pathways provided appropriate energy substrates are
present in the medium (19-22). Chart 1A illustrates the growth
kinetics of the glycolysis-deficient DS7 cells, the respirationdeficient GSK3 cells, and their parental O23 cells in medium that
contains both glucose and glutamina. Under these conditions,
the doubling time of the cells was 22, 15, and 12 h for DS7,
GSK3, and O23 cells, respectively. As expected, when glucose
was replaced by fructose, a low glycolytic substrate for these
cells, the growth of GSK3 cells ceased rapidly, while the doubling
time of 023 and DS7 cells was unaffected (14 and 20 h respec
tively; Chart 18). In this medium, GSK3 survival was maintained
for 4 h, and it decreased exponentially with time thereafter (Chart
2/4). Conversely, when oligomycin (0.5 /¿g/ml)was added to the
glucose containing medium, DS7 cells were rapidly inactivated
after a lag period of about 4 h, whereas the survival of the 023
cells was not affected (Chart 28). In fact, 023 and GSK3 cells
maintain exponential growth for at least 1 week in the oligomycincontaining medium (data not shown).
®
-5 11
li4!
ir«
®
l
t
t
Time at 44°
(hours)
2
Tu
at
3
44°(hour.)
Chart 3. Relative survival of 023 (•),DS7 (•).
and GSK3 (A) cells as a function
of time at 44°C. In A, exponentially growing cells were heat treated for the time
indicated, trypsinized, and plated at 37°Cfor colony formation. B, as in A, except
that the cells received a conditioning treatment of 30 min at 44°Cand were returned
to 37°Cfor 5 h before being exposed to 44°Cfor the time indicated.
-g u'
These phenotypic charateristics provide the opportunity to
evaluate the relative participation of glycolysis and respiration in
the maintenance of survival after hyperthermia. O23, DS7, and
GSK3 cells were heat treated for varying periods of time at 44°C,
u>
B
I
2
Time
3
(days)
4
5
6
and their survival was evaluated using a colony essay. As shown
in Chart 3/4, identical survival curves were obtained for the three
cell lines, independently of the energy producing pathway upon
which the cell relied. This indicates that the direct inactivation by
heat of glycolysis or respiration is not rate-limiting in hyperthermic
cell killing and that heated cells die owing to an initial perturbation
in an essential cellular function other than energy production.
1123456
Time
(days)
Chart 1. Growth kinetics of the glycolysis-deficient DS7 cells (•),
the respirationdeficient GSK3 cells (A), and the parental 023 cells (•)
plated into 25-cm2 dishes
in normal (A) or glucose-free fructose-containing medium (B). At the time indicated,
the cells were trypsinized and counted with an electronic cell counter. The medium
was changed every other day.
CANCER
RESEARCH
VOL. 46 JANUARY
1986
325
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ENERGY METABOLISM AND CELL RESPONSE TO HEAT
Indeed, if the Chinese hamster cells studied here were killed
through the direct thermal inactivation of their energy-producing
pathways, unless glycolysis and respiration are inactivated at
the same rate, it would follow that one of the mutants considered
(the one possessing the most thermosensitive pathway) would
be more heat sensitive.
The capacity of the 3 cell types to develop thermotolerance
and to be induced to synthesize HSP was also evaluated. O23,
DS7, and GSK3 cells were first subjected to a 30-min treatment
at 44°C and were then returned to 37°C for (a) 5 h and then
assessed for survival to a second treatment at 44°Cfor graded
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11. Sciandra, J. J., Subjeck, J. R., and Hughes, C. S. Induction of glucoseregulated proteins during anaerobic exposure and of heat-shock proteins after
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periods ot time or (b) for 0, 2, 4, 6, or 8 h and then incubated for
2 h in the presence of [35S]methionine. As shown in Chart 3B, a
prior heat treatment induces in the 3 cell clones a dramatic but
similar increase in cell resistance to the second treatment. Similar
results were obtained when a period of 10 h at 37°Cwas allowed
for the development of thermotolerance (data not shown). The
autoradiograms presented in Fig. 1 illustrate the kinetics of
induction of HSP synthesis in the cells. Heat induces in the 3 cell
lines a similar enhanced synthesis of 3 major polypeptides with
apparent molecular weights of 107,000, 70,000, and 68,000.
The HSP89 (Mr 89,000) was only slightly induced in these cells.
No differential response was observed between the cells in the
nature or quantity of HSP induced or in the kinetics of the
induction of the proteins. These results indicate that the partici
pation of respiration or glycolysis is not specifically required for
the induction of HSP and for the triggering and development of
thermotolerance and that thermotolerance does not result from
the protection of a particular energy-producing pathway.
The present study extends our previous observations which
showed that heat and various chemicals, including sodium arsenite, carbonyl cyanide m-chlorophenylhydrazone, oligomycin,
and antimycin A, induce HSP synthesis in chick fibroblasts
lacking a functional respiratory chain, as well as in control cells
(18). Heat induction of HSP synthesis was also observed in
respiration-deficient yeast (23). We suggest that thermosensitiv-
14. Mondovi, B., Strom, R., Rotilio, G., Agro, A. F., Cavaliere, R., and Rossi Fanelli,
A. The biochemical mechanism of selective heat sensitivity of cancer cells. I.
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mammalian cell inactivation: a study of the effects of glucose starvation and
an uncoupler of oxidative phosphorylation. J. Cell. Physiol., 707: 237-241,
1981.
17. Lunec, J. and Cresswell, S. R. Heat-induced thermotolerance expressed in the
energy metabolism of mammalian cells. Radiât.Res., 93: 588-597, 1983.
18. Landry, J., Chrétien,P., de Muys, J-M., and Moráis, R. Induction of thermo
tolerance and heat shock protein synthesis in normal and respiration-deficient
chick embryo fibroblasts. Cancer Res., 45: 2240-2247, 1985.
19. Franchi, A., Silvestre, P., and Pouysségur,A. A genetic approach to the role
of energy metabolism in the growth of tumor cells: tumorigenicity of fibroblast
mutants deficient either in glycolysis or respiration. Int. J. Cancer, 27: 819827,1981.
20. Pouysségur, J., Franchi, A., Salomon, J-C., and Silvestre, P. Isolation of a
Chinese hamster fibroblast mutant defective in hexose transport and aerobic
glycolysis: its use to dissect the malignant phenotype. Proc. Nati. Acad. Sci.
USA., 77: 2698-2701, 1980.
21. Zielke, H. R., Zielke, C. L., and Ozand, P. T. Glutamine: a major energy source
for cultured mammalian cells. Fed. Proc., 43: 121-125,1984.
22. Mckeehan, W. L. Glycolysis, glutaminolysis, and cell proliferation. Cell Biol. Int.
Rep.,6. 635-650,1982.
23. Undquist, S., DiDomenico, B., Bugaisky, G., Kurtz, S., Petko, L., and Sonoda,
S. Regulation of the heat shock response in Drosophila and yeast. In: M. J.
Schlesinger, M. Ashbumer, and A. Tissieres (eds.), Heat Shock from Bacteria
to Man, pp. 167-175. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory,
1982.
24. Gerweck, L. E., Dahlberg, W. K., Epstein, L. F., and Shimm, D. S. Influence of
nutrient and energy deprivation on cellular response to single and fractionated
heat treatments. Radiât.Res., 99: 573-581,1984.
25. Calderwood, S. K., Bump, E. A., Stevenson, M. A., Van Kersen, I., and Hahn,
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ity, thermotolerance development, and HSP induction do not
depend on an initial direct interaction of heat with the metabolic
pathways responsible for the production of cellular energy. How
ever, it cannot be ruled out that a lack of energy is the final
cause of hyperthermic cell death and that a drop in the cellular
energy level is part of the cascade of events that lead to the
induction of thermotolerance and HSP. In this case, the present
results would favor the hypothesis that the initial signal is gen
erated through a heat-induced cell alteration that results in an
enhanced consumption of cellular energy or in the indirect per
turbation of the regulatory mechanism of energy production,
rather than through a direct physical interaction of heat with
glycolysis or respiration. Such an indirect interaction of heat with
energy metabolism would explain the increased thermosensitivity
of energy depleted cells (24, 25).
ACKNOWLEDGMENTS
We are indebted to Dr. Jacques Pouysségurfor the gift of his clones O23, DS7,
and GSK3.
CANCER
RESEARCH
VOL.
46 JANUARY
1986
326
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ENERGY METABOLISM AND CELL RESPONSE TO HEAT
-
G LYCOLYSIS-
RESPIRATIONCONTROL
DEFICIENT
DEFICIENT
»
I
C
02468
C
O
2
4
68
C
O 2
4
6
8
Fig. 1. Induction of HSP in O23 (control), GSK3 (respiration-deficient),and DS7 (glycolysis-deficient)cells. The cells were incubated for 2 h in the presence of
[œS]methioninebeginning at various times (indicated ¡n
h below each lane) after they received a 30-min treatment at 44°C.Total proteins were extracted and processed
for electrophoresis on a 10% polyacrylamidegel slab in the presence of sodium dodecyl sulfate. The whole procedure has been described in detail elsewhere (4,5,18).
A constant amount of protein was placed on the top of each lane. C, control protein synthesis pattern of non-heatedcells. The arrowheads indicate the position of HSP;
from fop to bottom, M, 107,000, 89,000, and 70,000-68,000.
CANCER
RESEARCH
VOL. 46 JANUARY
1986
327
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1986 American Association for Cancer Research.
Hyperthermia-induced Cell Death, Thermotolerance, and Heat
Shock Proteins in Normal, Respiration-deficient, and
Glycolysis-deficient Chinese Hamster Cells
Jacques Landry, Stéphanie Samson and Pierre Chrétien
Cancer Res 1986;46:324-327.
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