[CANCER
RESEARCH
45, 3816-3824,
August 1985]
Induction of Heat Shock Protein Synthesis in Murine Tumors during the
Development of Thermotolerance1
Gloria C. Li2 and Johnson Y. Mak
Radiation Oncology Research Laboratory, Department of Radiation Oncology, The University of California, San Francisco, California 94143
ABSTRACT
The function of one or more heat shock proteins (HSPs) may
be to confer protection of cells against thermal damage. We
examined the induction kinetics of thermotolerance and the
synthesis of HSPs in murine tumor models. Squamous cell
carcinomas (SCC VII/SF) or radiation-induced fibrosarcomas
(RIF) were implanted in the flanks of C3H mice. These flank
tumors were first exposed to an elevated temperature (41°45°C)for a fixed duration, for example, 43°Cfor 15 min. Some
of the tumors were excised immediately, and tumor cell suspen
sions were made. The other mice with tumors were returned to
the cages and left undisturbed for various times up to 72 h
before being sacrificed. Again, tumors were then removed and
tumor cell suspensions were prepared. These tumor cells were
either challenged with a second heat treatment at 45°Cin vitro
or labeled with [^SJmethionine at 37°Cin vitro. The tumor cell
survival after the combined heat treatments was measured using
the in vitro cloning assay. The cellular proteins were analyzed by
one- or two-dimensional gel electrophoresis. We found that mild
heat shock induced thermotolerance in murine tumors, a result
consistent with those of others. The kinetics of induction and
decay of thermotolerance depended on the temperature and
duration of the priming treatment. Mild heat shock also enhanced
the rate of synthesis and accumulation of some HSPs during the
development of thermotolerance. For example, after an initial
treatment at 43°C for 15 min, the rates of synthesis of HSPs
with molecular weights 68,000,70,000, and 87,000 were greatly
enhanced in SCC VII/SF tumors when compared to unheated
controls. Qualitatively similar results were seen with radiationinduced fibrosarcoma tumors. The rate of synthesis of M, 68,000
to 70,000 HSPs reached maximum value (300% of control value)
2 to 4 h after heat shock and decreased to the control value 6
to 24 h later. On the other hand, the rate of synthesis of actin, a
major structural cellular protein, remained relatively constant
throughout the 72 h of experiments. We then determined the
relationship between the synthesis and accumulation of these
HSPs and the expression of thermotolerance in murine tumors
after a priming heat treatment. The data indicate that the levels
of Mr 68,000 to 70,000 HSPs correlate well with thermotolerance.
Thus, it appears likely that the measurement of levels of these
HSPs can be used as an assay to determine the degree of
thermotolerance in tissues during fractionated hyperthermia as
applied in the clinic.
INTRODUCTION
Mammalian cells, when exposed to a nonlethal heat treatment,
1This work was supported by NIH Grant CA-31397.
2 To whom requests for reprints should be addressed, at Radiation Oncology
Research Laboratory, CED-200, Department of Radiation Oncology, University of
California, San Francisco, CA 94143.
Received 9/7/84; revised 4/3/85; accepted 4/18/85.
CANCER
RESEARCH
have the ability to acquire a transient resistance to a subsequent
heat challenge. This phenomenon has been termed thermotol
erance (1-3). The induction of thermotolerance, its development,
and its subsequent decay, both in vitro and in vivo, have been
studied by many investigators using various cell lines (1, 3, 45), various rodent tumors (6-12) and normal tissues (13-19).
Even though different endpoints were used in these studies and
the experimental designs sometimes did not allow for differentia
tion between recovery from the heat-induced sublethal damage
and development of thermotolerance, the results from the in vivo
studies were qualitatively similar to those from in vitro studies.
The molecular mechanisms of thermotolerance are not well
understood. It has been suggested that HSPs3 may play a role
that is related to heat-induced thermal protection in Drosophila
and yeast (20, 21). HSPs are a family of proteins the synthesis
of which is either induced or enhanced by heat shock or other
environmental stresses (22). This group of proteins is induced in
a wide variety of biological systems ranging from Drosophila to
yeast and mammalian cells (20-27). Many investigators, in the
past few years, have performed experiments to determine the
relationship between thermotolerance and HSP synthesis using
various mammalian cell lines. So far, most studies show a good
temporal relationship between the development of thermotoler
ance and enhanced synthesis of HSPs (25,26,28-30);
although
there are reports that suggest that correlation is not universal
(30, 31 ). Recently, we have examined the quantitative relation
ship between cell survivals after a 45°C,45-min heat treatment
and the levels of HSPs
hamster fibroblasts and
(32). These results show
of HSP with a molecular
cells' thermal sensitivity.
in transient thermotolerant Chinese
in their stable heat-resistant variants
a good correlation between the levels
weight around 70,000 (HSP 70) and
During decay of tolerance when the
concentration of HSP 70 decreases, cells also become much
more sensitive to thermal stress. These data indicate that the
level of HSP 70 appears to be a good predictor of thermal
response in vitro. However, no similar data are available from
animal tumor models.
We report here on experiments designed to study the effects
of hyperthermia (41 °-45°C)on the induction of thermotolerance
and on the profiles of protein synthesis in murine tumor models,
a squamous cell carcinoma (SCC VII/SF), and a RIF. Specifically,
we examined whether the synthesis of HSPs can be induced or
enhanced in murine tumors after a nonlethal heat treatment. We
also investigated the kinetics of the development of thermotol
erance by doing survival experiments and the time patterns of
synthesis of HSPs and other cellular proteins during the devel
opment of thermotolerance using gel electrophoresis techniques.
Finally, we determined the relationship between HSP synthesis
3The abbreviations
used are: HSP, heat shock protein; HSP 70, heat shock
protein of molecular weight 70,000 (other heat shock proteins are similarly desig
nated); MEM, minimal essential medium; SCC VII/SF, squamous cell carcinoma;
RIF, radiation-induced fibrosarcoma; SDS, sodium dodecyl sulfate.
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HEAT SHOCK PROTEINS
AND THERMOTOLERANCE
and thermotolerance. If human tumors behave as these murine
tumors do, then our data suggest the possibility of using a
specific HSP (or a family of HSPs) as predictors for thermal
response in tumors during fractionated hyperthermia.
MATERIALS
AND
as in the heat experiment. Immediately, 4 or 24 h after the anesthesia,
animals were sacrificed, and the tumor cells were exposed to the 45°C
heat treatment. As shown in Chart 3, anesthesia did not cause a
significant increase in tumor cell survival after 45°Cheat challenges.
Labeling and Gel Electrophoresis. The labeling experiments were
performed in vitro, parallel to the survival experiments, but involved only
the initial heat treatment. After the tumors had been exposed to elevated
temperature, the tumor-bearing animals were allowed to recover. At
METHODS
Animal Tumor System and Hyperthermic Treatment. The tumors
studied were the squamous cell carcinoma (SCC VII/SF) and RIF tumors,
both syngeneic in C3H mice. The characteristics of these tumors have
been described previously (33, 34). Tumors were implanted into flanks
of C3H mice by subcutaneous inoculation of 106 viable tumor cells and
various times after the first heat treatment, usually from 0 to 72 h, the
tumors were excised, and a single cell suspension was prepared as
described above. Tumor cells were resuspended in methionine-free MEM
and labeled with ["SJmethionine (specific activity, 1200 Ci/mmol; Amersham) at concentrations of 20 to 40 //Ci/ml for 1-dimensional gels or 200
to 400 /iCi/ml for 2-dimensional gels. After appropriate lengths of labeling
at 37°C(usually 2 h for 1-dimensional gel and 4 h for 2-dimensional gel),
were used for experiments approximately 10 to 12 days later when the
tumor size reached -500 cu mm (8 to 10 mm, longest diameter). The
cells were washed twice with ice-cold phosphate-buffered
saline and
lysed in SDS-sample buffer (26, 32) for 1-dimensional analysis or in
isoelectric focusing-sample buffer for 2-dimensional analysis (36). One-
average tumor weight then was 400 to 600 mg. At the time of heat
treatment, animals were anesthetized with Diabutal (60 mg/kg). To heat
the flank tumor in vivo, the tumor-bearing mouse was secured inside a
"boat" which was modified from a T-75 Falcon tissue culture flask. The
dimensional analysis in the presence of SDS was also performed as
described (37), except that the running gels contained 12.5% acrylamide.
Two-dimensional gel electrophoresis was performed as described by
O'Farrell (36). Following electrophoresis, gels were stained with Coo-
top side of the T-flask was removed completely, and the bottom side of
the T-flask had a hole of 2 cm in diameter. The flank tumor was pulled
gently through this opening and immersed directly into the circulating hot
water bath stabilized to within ±0.05°Cof preset temperatures. Tem
massie blue G-250 in 3.5% perchloric acid, destained in 7% acetic acid,
dried, and autoradiographed on Kodak SB-5 X-ray film. Care was taken
peratures of the tumor core, mouse rectum, and of the hot water bath
were monitored routinely.
The tumor core and rectal temperature were initially low due to
anesthesia but rose gradually when the tumor was immersed into the
water bath. The tumor core temperature increased much more rapidly
than the rectal temperature. The intratumor temperature stabilized within
a few minutes to approximately 0.5°Cbelow the water bath temperature.
The water bath was therefore adjusted to 0.5°Cabove the desired tumor
to remain within the linear exposure range of the film.
Autoradiograms of 1-dimensional gels were quantitated
with protein synthesized in control tumors. During the development of
thermotolerance, the relative rate of HSP synthesis was expressed as a
percentage of the incorporation rate for the specific protein in heatshocked cells with respect to the nonheated controls. The incorporation
rate of a specific protein was expressed as percentage of total [MS]
methionine-labeled
proteins. This was determined
by the ratio of the
integrated area under each protein peak to the total area under the scan
that included all protein bands in the gel lane. Although, on 2-dimensional
gel, we can clearly separate the M, 68,000 to 70,000 proteins into at
least 2 separate spots, separation on 1-dimensional gel for individual
quantification of M, 68,000 and 70,000 protein is difficult. We therefore
analyzed the M, 68,000 to 70,000 region as a composite protein peak
and referred to it as HSP 70 for simplicity.
Experimental Protocols. A schematic flow diagram indicating our
experimental protocols is shown in Chart 1. After the first heat exposure
in situ, some of the tumors were excised immediately (0 h point), and a
single tumor cell suspension was made. The other mice with preheated
flank tumors were left undisturbed for various times up to 72 h before
being sacrificed. The tumors were then excised, and a tumor cell sus
pension was made. The prepared tumor cell suspension was divided into
2 parts; one-half was used for survival studies, and the other half were
were then returned to the cages for times up to 72 h before being
sacrificed. Tumors were then excised for cell survival experiments and
protein analysis.
Cell Survival Assay. Treated tumors were excised, pooled, weighed,
minced, and suspended in phosphate-buffered saline containing neutral
protease (1 mg/ml; Boehringer Mannheim). Cell suspensions were fil
tered, centrifuged, and resuspended in MEM. Tumor cell concentration
was calculated by counting the cells using a hemocytometer. Cells were
then serially diluted in MEM supplemented with 20% heat-inactivated
calf serum, and a known number of cells was plated on 60-mm Petri
used for labeling experiments and protein analysis. For survival studies,
known numbers of tumor cells were plated and incubated at 37°Cfor 2
dishes. Each dilution was plated on 2 to 4 dishes. After 2 h incubation
at 37°Cto allow good cell attachment, these cells were then challenged
by a second heat treatment at 45°C in vitro. The in vitro heating was
h to allow good cell attachment. These monolayers of cells were then
challenged by a second heat shock at 45°Cfor 0 to 45 min in vitro (D2).
carried out by exposing these monolayers of cells to hot water bath in a
specially designed incubator (35). The pH of the medium was maintained
between 7.2 and 7.4 by a regulated flow of a CO2 and air mixture. After
the second heat treatment, cells were returned to 37°Cincubation for 8
The tumor survival after the combined heat treatment was determined
using cell survival assay.
For protein analysis, the other half of the prepared tumor cell suspen
sion was resuspended in methionine-free medium. A known number of
cells, usually around 0.5 to 1.0 x 106 cells, was labeled with [MS]
methionine for 2 h at 37°C. The tumor cellular proteins were then
to 10 days. The clones were fixed, stained, and counted. The cloning
efficiency of unheated control tumor cells was 36 ±10 (SD) % for SCC
VII/SF tumors and 49 ± 10% for RIF tumors, respectively. Surviving
fraction was defined as cloning efficiency of cells from treated tumor
divided by the cloning efficiency of cells from control tumors without both
first and second heat treatment. Heat survival curves for control non-
analyzed by 1- to 2-dimensional gel electrophoresis.
We want to stress the point that the first heating (D,) was performed
in vivo. Tumors were then maintained in situ for various times until the
second treatment of either heating or labeling. To minimize the compli
cation caused by heat-induced modification of blood flow and microen-
thermotolerant tumors were from animals treated identically but without
application of the first in vivo heat treatment.
A control experiment was performed to test the effect of anesthesia
on the induction of thermotolerance. Control animals were anesthetized
RESEARCH
with an LKB
model 927 laser densitometer. The enhanced syntheses of HSPs were
identified by comparing proteins synthesized in heat-shocked tumors
temperature. All temperatures mentioned in this paper refer to the
intratumoral temperature, and all heating intervals include the transient
warmup time.
To study the kinetics of development of thermotolerance and synthesis
of HSPs, flank tumors were first exposed to elevated temperature (41 °C45°C)as described. Following heating, some of the animals were sacri
ficed immediately (0 h point). The other animals were kept warm (36°37°C) during recovery from anesthesia. These tumor-bearing animals
CANCER
IN MURINE TUMORS
vironment in tissues, the second heat treatment or the labeling of tumor
cells was performed in vitro.
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HEAT SHOCK PROTEINS
AND THERMOTOLERANCE
RESULTS
Induction and Decay of Thermotolerance in Murine Tumors
SCC VII/SF Tumors. The kinetics of induction and decay of
thermotolerance in SCC VII/SF tumors after an initial exposure
(D,) at 41 °Cfor 60 min, 41.5°C for 40 min, or 42°Cfor 40 min
are shown in Chart 2. If the initial treatment was at 41 °Cor
42°C, thermotolerance had already developed to a significant
IN MURINE TUMORS
treatments was increased up to 24 h. The kinetics of the decay
of thermotolerance after 41 °Cor 42°C initial heating are also
demonstrated in Chart 2. If the initial treatment was at 41 °Cfor
60 min, tolerance decayed almost completely by 72 h. The 45°C
survival curves for the 72-h preheated tumors were indistinguish
able from those of the control tumor cells.
If the first heat treatment was at 43°Cor a higher temperature
degree within 2 to 4 h after the initial heating. By 6 h, the
tolerance was essentially at its maximum. This was evidenced
by the increase in survival from 10~4 of the control tumor cells
to 10~1 of the 4-h thermotolerant tumor cells after a heat chal
lenge at 45°C for 45 min. In addition, there was only a minor
(Chart 3), some time in situ at normal body temperature was
required to allow the development of thermotolerance. Tolerance
was at its maximum between 6 to 24 h after the initial heating.
Thermotolerance lasted longer; by 72 h after the first heat dose
(D,), there was still a significant degree of thermotolerance in the
tumors. This was evidenced by the much higher values of the
45°C survival of the 72-h tolerant cells when compared to the
increase in survival values if the interval between the 2 heat
control tumors with no preheat treatment (Chart 3).
SECOND
HEAT EXPOSURE
DATA ANALYSIS
IN VITRO
45°C,0-45
IN VITRO
MIN
CELL SURVIVAL ASSAY
TUMOR CELL SUSPENSION
FIRST HEAT EXPOSURE
\N SITU
43°C, 15 MIN
DATA ANALYSIS
FLANK TUMORS
1. AUTORADIOGRAM
2. DENSITOMETER TRACING
Chart 1. Schematic diagram showing the
sequence of experimental protocols. The sur
vival studies of the kinetics of development and
decay of thermotolerance were performed in
parallel with the protein analysis using the same
groups of tumors. The first heat treatment and
the subsequent recovery were in situ. The sec
ond heat treatment and the labeling were per
formed in vitro using tumor cell suspensions.
3. QUANTIFICATION OF THE
RATE OF PROTEIN
SYNTHESIS
48
'•
72
CONTROLO
(a)
(b)
41.5°C, 40 MIN
41°C, 60 MIN
105
30
16
TIME
AT
15
45
TIME
45°C ( MIN )
30
AT
45°C ( MIN )
Chart 2. Cell survival from SCC VII/SF tumors receiving a first exposure (D,) in
vivo (listed) followed by graded second treatments (D2) at 45°C in vitro. Times
between D, and tumor excision in h are indicated. Controls are tumor cells receiving
D2 treatment only. Near-maximum levels of tolerance are seen 4 h after Dv
CANCER
RESEARCH
45
45
TIME
AT
45°C ( MIN )
Tolerance decays almost completely 72 h after D,. The variations shown on the
surviving fractions on 0 time at 45°Crepresent the tumor cell survivals at various
times after the first priming heat treatment, a, D, = 41 °Cfor 60 min; 6, D, =
41.5°Cfor 40 min; c, D¡= 42°Cfor 40 min. Bars, SD.
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HEAT SHOCK PROTEINS
AND THERMOTOLERANCE
IN MURINE TUMORS
Z
O
K
O
OC
LL
o
e
10 -
cr
co
C)
CONTROL
44.5°C, 10 MIN
15
TIME
AT
45°C ( MIN )
TIME
AT
45°C ( MIN )
Chart 3. Cell survival from SCC VII/SF tumors receiving a first exposure (D,) in
vivo (listed) followed by graded second treatments at 45°Cin vitro. Tolerance is at
TIME
30
AT
45
45°C ( MIN )
not cause a significant increase in cell survival (see control, 4 h; a). Anesthesia
given 24 h earlier also does not cause significant increase in cell survival, a, D, =
43°Cfor 15 min; b. Di = 44°C for 15 min; c, D, = 44.5°C for 10 min. (Detailed
its maximum between 6 and 24 h after D,. There is still a significant degree of
tolerance in the tumors 72 h after D, treatment. Anesthesia given 4 h earlier does
legend as in Chart 2). Bars, SD.
RIF Tumors. Similar experiments as those described above
were carried out to examine the induction and decay of thermotolerance in RIF tumors. The RIF tumors were first exposed
to 41.5°C for 40 min or 44°Cfor 15 min (Di). At 2 to 72 h later,
tumors were excised, and a single cell suspension was prepared.
A known number of cells was plated and was subsequently
challenged by graded treatments at 45°C (D2). Cell survivals
plotted as a function of D2 are shown in Chart 4. Near-maximum
levels of tolerance are seen 4 h after Di treatment.
72
Kinetics of Synthesis of Heat Shock Proteins during the
Development of Thermotolerance in Murine Tumors
(a
SCC VII/SF Tumors. Studies on the kinetics of HSP synthesis
during the development of thermotolerance in SCC VII/SF tumors
were done in parallel to the survival studies. In most cases, the
same tumors from the above treatment groups used for survival
studies were also used for the labeling experiments.
Autoradiograms of SDS-polyacrylamide slab gels of [35S]methionine-labeled proteins from control and heated SCC VII/SF
tumors and the densitometer tracings from the autoradiograms
are presented in Fig. 1 and Chart 5. It is demonstrated clearly
after an initial treatment of 43°C for 15 min that the rates of
synthesis of HSPs with molecular weights 68,000, 70,000, and
87,000 were greatly enhanced when compared to the unheated
controls. It is also shown in Fig. 1 that anesthesia alone did not
cause enhanced synthesis of HSPs. Similar time patterns of
protein synthesis were observed at other heating temperatures
(Fig. 2). These HSPs were not simply novel components of heatstressed tumor cells, since most of the proteins were also
constitutively expressed in nonheated controls. As already
CANCER
RESEARCH
)
CONTROL
41.5°C, 40 MIN
30
TIME
AT
45°C ( MIN )
15
TIME
30
AT
45
45°C ( MIN )
Chart 4. Cell survival from RIF tumors receiving a first exposure (Di) in vivo
followed by graded second treatments (D2) at 45°Cin vitro, a, D, = 41.5°C for 40
min; b, D, = 44°Cfor 15 min. (Detailed legend as in Chart 2). Bars, SD.
pointed out in "Materials and Methods," the M, 68,000 to 70,000
region was analyzed as one composite protein peak and referred
to as HSP 70. The rates of synthesis in unheated tumors were
6.8 ± 1.8%, 3.2 ± 0.9%, and 19.4 ± 1.0% of total protein
synthesis for HSP 70, HSP 87, and actin, respectively. In Chart
6, the relative rates of synthesis, expressed as a percentage of
nonheated controls, of individual HSPs and actin are plotted as
a function of recovery time in situ after the initial heat treatment.
We found that the rates of synthesis of the HSP 70 and the HSP
87 reached maximum 2 to 4 h after heat shock and returned to
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HEAT SHOCK
PROTEINS
AND THERMOTOLERANCE
4
IN MURINE TUMORS
CONTROL
4HR
CONTROL
3
2
1
O
4
HEATED,
O HR
HEATED,
6HR
A
3
2
W
Z
111
t-
Chart 5. Densitometer tracings from the autoradiogram shown in Fig. 1. Molecularweights
(x10~3) are shown at the bottom. Actin (M,
Z
~
43,000) is indicated by A.
LU
1
O
4
HEATED,
24 HR
HEATED,
A
2HR
3
LU
OC
2
1
O
4
HEATED,
48 HR
3
2
1
O
110
87 70 59
68
43
34
26
MOLECULAR
110
87
70 59
68
43
34
26
WEIGHT
control values by 24 h. On the other hand, the rate of synthesis ment, the rate of synthesis for M, 66,000 to 70,000 HSP was 3for actin, a major structural cellular protein, remained relatively- fold higher than that of the controls.
constant throughout the 72 h of the experiments. Examining the Effect of Tumor Dissociation on the Thermal Response of
proteins by 2-dimensional gel electrophoresis, we can identify at Control and Thermotolerant Cells
least 2 major polypeptides in the M, 68,000 to 70,000 region
The effect of trauma, caused by the experimental procedures
(Fig. 3).
RIF Tumors. Densitometertracingsfrom the autoradiogram used to prepare a single cell suspension from SCC VII/SF
of a SDS-polyacrylamide slab gel of [35S]methionine-labeledpro
tumors, on the thermal response of control or thermotolerant
teins from control and heated RIF tumors are shown in Chart 7.
Again, it is demonstrated clearly that after an initial treatment of
44°Cfor 15 min, the rate of synthesis of HSPs with molecular
cells, was examined. Cells from control tumors, tumors immedi
ately after a 43°C,15-min in vivo priming dose, or thermotolerant
tumors (4 h after the 43°C,15-min priming dose) were challenged
by a 30-min, 45°Cin vitro heat treatment at various times after
37°Cincubation of the single tumor cell suspension preparation.
weights of 66,000,68,000,70,000, and 87,000 were significantly
enhanced. For example, 2 to 4 h after the 44°C,15-min treat
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HEAT SHOCK PROTEINS
-,.
AND THERMOTOLERANCE
IN MURINE TUMORS
The results are shown in Chart 8. The control unheated and
thermotolerant tumors had very different survival responses. For
control unheated tumor, the survival value was 10~4 if the 45°C
--
heat challenge was given immediately after tumor dissociation;
if the 45°Cheating were given 2 h later, the survival was 10~2,
a 100-fold increase in survival. On the other hand, for the cells
obtained from thermotolerant tumors (i.e., 4 h after the 43°C
priming heating in vivo), the survival after a 45°C, 30-min heat
87 ~-
treatment given immediately after tumor dissociation was 5 x
10~2. If the 45°Cheating was given 2 h later, the survival was 2
x 10~1, only a 4-fold increase. When the survival of the tumor
cells obtained from the tumors immediately after the 43°C in
vivo priming dose is plotted as a function of 37°C incubation
before the 45°C heating, the data in Chart 8 (O h curve) dem
onstrated clearly that thermotolerance can be developed in vitro
after tumors were preheated in vivo. In this case, tolerance
reached its maximum 3 to 4 h after the first heat challenge.
DISCUSSION
C CA 0
Fig. 1. Autoradiogram
2
4
of a SDS-polyacrylamide
6 24 48
slab gel of [^SJmethionine-
labeled proteins. The enhanced synthesis of M, 68,000, 70,000, and 87,000 HSPs
in cells from heated (43°C, 15 min) SCC VII/SF tumors is evident. C, unheated
controls. C»,unheated control recovered from anesthesia given 4 h earlier. 0 to 48,
recovery time (h) in situ after D, and before In vitro labeling. Molecular weights
(x10~3) are shown at left. Actin, identified by molecular weight (M, 43,000), is
indicated by A.
O
OC
3
43° 41.5°
3
4.5 4.5
6 6
16
There is considerable experimental evidence showing that
thermotolerance can be induced in tumors and can thus modify
the tumor response to fractionated hyperthermia. For example,
Mäheref al. (7) examined the thermal resistance in a sponta
neous murine fibrosarcoma tumor implanted into the feet of C3Hf
mice. Urano eÃ-al. (11) compared the response after multiple
hyperthermia and the kinetics of thermotolerance between a
spontaneous C3Hf mouse mammary carcinoma and a chemically
induced fibrosarcoma. Nielsen and Overgaard (8) also examined
the effect of temperature and time of preheat treatment in a C3H
mammary carcinoma. Most recently, Rofstad and Brustad (10)
studied the kinetics of the development of human melanoma
xenograft. Rhee et al. (9) assayed the cell survival of SCK
mammary carcinoma cells after fractionated hyperthermia at
43.5°C. Even though different end points, such as tumor growth
time analysis, tumor control dose, or in vitro cell survival, were
used in these studies, the data agreed well. All investigators
found that thermotolerance was induced by a short priming heat
dose, developed rapidly, reached maximum within 24 h after the
first treatment, and decayed slowly. The magnitude of thermo
tolerance to the second heat treatment increased with increasing
priming dose. If the preheating time was increased at a constant
preheating temperature, the degree of tolerance and the time
interval required to allow maximal development was also in
creased.
In this study, in addition to measuring cell survival, we asked
specific questions about protein synthesis in murine tumors
during fractionated hyperthermia. Can synthesis of HSP 70 be
induced in tumors? Is HSP 70 synthesized in unheated controls?
What is the relationship between HSP synthesis and thermotol
erance? Can the concentration of HSP 70 be used to predict the
thermal response of tumors or to predict the degree of residual
thermotolerance during fractionated hyperthermia? Our data
showed that mild heat shock in the temperature range of 41 °C45°C induced thermotolerance in murine tumors, results con
16
43° 41.5" 43° 41.5°43° 41.5°43° 41.5°
beled proteins in cells from SCC VII/SF tumors after an initial Di exposure in vivo
at 43°Cfor 15 min or 41.5°C for 40 min. C, unheated controls. 0 to 76, recovery
(h) time In situ after 0, and before in vitro labeling. Molecular weights (x10~3) are
sistent with those of others. The kinetics of induction and decay
of thermotolerance depended on the temperature and duration
of the priming heat treatment. If the initial treatment temperature
was at 41 °or 42°C, thermotolerance almost fully developed 2
shown at left. Actin (M, 43,000) is indicated by A.
to 4 h after the initial treatment and decayed almost completely
Fig. 2. Autoradiogram
of SDS-polyacrylamide
slab gel of ["SJmethionine-la-
CANCER
RESEARCH
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HEAT
SHOCK
PROTEINS
AND
THERMOTOLERANCE
IN MURINE
TUMORS
O
ce
o
o
l
Charte. Kinetics of HSP synthesis during
the development of thermotolerance. Relative
rates of synthesis, expressed as a percentage
of nonnested controls, of individual HSPs (M,
68,000 to 70,000 and M, 87,000) and a major
cellular structural protein, actin, are plotted as
a function of recovery time in situ after the initial
D, treatment. As pointed out in "Materials and
Methods," the M, 68,000 to 70,000 region is
l
_1
I
L
l__
I
I
_J
I
I
lili
co
co
Hi
X
co
analyzed as one composite protein peak. 9. D,
= 41 °Cfor 60 min; O, D, = 42°C for 40 min;
A, D, = 43°Cfor 15 min; and A, Dt = 44.5°C
for 10 min.
o
oc
o.
.
o
LU
H
IX
O 4
8 12 16 2024
TIME
by 72 h. If the initial treatment temperature was 43°Cor higher,
a few h at 37°Cwas required before thermotolerance was fully
Synthesis of HSP
Accumulation of
HSP8
Thermotolerance
unstressed
+"
Time after hyperthermic shock:
Oh
2h
4h
6h
1 day 2 days
+
—
Determined on 1-dimensional gel stained with Coomassie blue G-250.
' —, undetectable; +, barely detectable; ++, detectable; +++, easily detect
able.
h for this sensitivity to disappear, as evidenced by a 100-fold
increase in survival. Under similar conditions, only a very minor
difference in thermal sensitivity was seen for cells obtained from
thermotolerant tumors. When the survival of the cells obtained
from the tumors immediately after the 43°Cin vivo priming dose
was plotted as a function of 37°C incubation before the 45°C
RESEARCH
37°C ( HR )
Normal
dose, thermotolerance lasted longer; by 72 h, a substantial
degree of thermotolerance remained.
It is of interest to point out that, when tumors were in their
thermotolerant state, very probably they were also more resist
ant to the trauma caused by the sequential experimental proce
dures used to prepare tumor single-cell suspension, i.e., from
excising the tumors, mincing the tissues to cell dissociation. As
demonstrated in Chart 8, the control tumor cells heated imme
diately after the preparation of single cell suspensions were very
sensitive to the 45°C,30-min heat treatment, and it took 2 to 4
CANCER
AT
72
Table 1
Synthesis and accumulation of HSP 70 and thermotolerance in murine tumors
alter 43°C hyperìhermia
expressed. Thermotolerance was at its maximum between 6 to
24 h after the initial heating. In contrast to 41°or 42°Cpriming
heating (Chart 8, 0 h curve), it is demonstrated clearly that
thermotolerance also developed in vitro after tumor cells were
preheated in vivo. The expression of tolerance reaches its max
imum 3 to 4 h after the first heat challenge. The kinetics of
development of thermotolerance in vitro is similar to the kinetics
48
of thermotolerance in vivo.
During the development of thermotolerance, the rates of syn
thesis of some of the high-molecular weight HSPs, specifically
the HSP 70 and the HSP 87, were enhanced. Two-dimensional
gel analysis indicated that the M, 68,000 to 70,000 region con
sisted of at least 2 major polypeptides. These HSPs are not
simply novel components of heat-shocked tumors, since they
are also expressed constitutively in nonheated controls. The rate
of synthesis of HSP 70 reached maximum 2 to 4 h after the
priming heat dose, returning to the control rate by 24 h. The rate
of synthesis of HSP 87 was also enhanced shortly after the initial
heat treatment and returned to the control rate by 24 h. On the
contrary, 24 h after initial heating, thermotolerance was at its
VOL. 45 AUGUST 1985
3822
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HEAT SHOCK
PROTEINS
AND THERMOTOLERANCE
IN MURINE TUMORS
10e
io-1
z
g
i-
0
CONTROL
ü
CO
z
W
z
o
>
P
<
_i
1C
uu
rr
3
DC
D
CO
10
'4
IN VIVCHN VITRO
43°C.15 MIN
— 45°C.30MIN
87
68
70
43
MOLECULAR
WEIGHT
Chart 7. Densitometer tracings from the autoradiogram of an SDS-polyacrylamide slab gel of [^Sjmethionine-labeled proteins from RIF tumors after an initial
D, exposure at 44°Cfor 15 min. Molecular weights (x10~3) are shown at bottom.
Actin, identified by molecular weight (M, 43,000), is indicated as A. The rates of
synthesis of M, 66,000, 68,000, and 70,000 HSP are significantly enhanced 2, 4,
even 6 h after the D, treatment, but they gradually returned to the control rate by
24 h.
maximum; it only gradually decayed, and it returned to the control
value at least 72 h later. Thus, the kinetics of the rate of enhanced
synthesis of HSP after a single heat treatment do not correlate
with the decay of thermotolerance.
In Table 1, we present the rate of synthesis, the accumulation
of M, 68,000 to 70,000 HSP, and the expression of thermotol
erance in SCC VII/SF tumors after a 43°C, 15-min heat treat
ment. The data indicate that the levels of HSP 70 but not the
rate of synthesis, correlate well with the expression of thermo
tolerance. The results agree well with our previous observation
using tissue culture cell lines (32). Thus, it appears likely that the
measurement of the levels of HSP 70 can be used as an assay
to determine the degree of thermotolerance or even the thermal
response of tumor tissues during fractionated hyperthermia as
applied in the clinic.
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CANCER
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CONTROL
HEATED
87
X
87
v '•
\
x x
70
••
•
.
Fig. 3. Autoradiogram of a 2-dimensional gel showing the increased synthesis of the M, 68,000 and 70,000 HSPs in SCC VII/SF tumors after 42°Chyperthermia.
Tumor cells from control and heated (42°C,40-min) tumors were labeledwith [MS]methioninefor 4 h after D,. Total cellularextracts were analyzed by isoelectricfocusing
in the first dimension (from right to left) and SDS-polyacrylamidegel electrophoresis in the second dimension(from top to bottom). A, actin; V, vimentin. Arrows indicate
the M, 70,000 HSP family in heated and in control unheated cells.
CANCER
RESEARCH
VOL. 45 AUGUST 1985
3824
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1985 American Association for Cancer Research.
Induction of Heat Shock Protein Synthesis in Murine Tumors
during the Development of Thermotolerance
Gloria C. Li and Johnson Y. Mak
Cancer Res 1985;45:3816-3824.
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