(CANCER RESEARCH 49, 1492-1496, March 15, 1989]
Effects of Heat Shock Proteins (Afr 70,000) on Protein and DNA Synthesis at
Elevated Temperatures in Vitro1
Nahid F. Mivechi2 and Peter D. Ogilvie
Department of Radiation Research, City of Hope National Medical Center, Duarte, California 91010
ABSTRACT
The protective effect of M, 70,000 heat shock protein (HSP-70) during
thermotolerance has been previously observed. However, it is not known
what cellular processes or components may be protected by this protein
during the tolerance state. In the studies reported here, the protective
effects of purified HSP-70, the nonspecific heat-stable proteins fetuin
and trypsin inhibitor (ovomucoid), and other proteins and agents such as
bovine serum albumin, D2O, or glycerol on protein and DNA synthesis
during heating were investigated in vitro. In vitro protein synthesis at
30, 40, and 42°Cwas measured by globin mRNA translation. Protein
synthesis was inhibited 40 to 70% when incubated for 60 min at 40 and
42°C.However, protein synthesis was protected when either fetuin or
ovomucoid was present during protein synthesis at elevated temperatures.
The protection was concentration dependent. The HSP-70 purified from
Chinese hamster (HA-1) cells was also able to confer protection to the
translation system, but at much lower concentrations than either fetuin
or ovomucoid. Other proteins, such as bovine serum albumin, or other
agents, such as D2O or glycerol which are known protectors of cellular
survival during heating, did not protect the translation system.
Similar experiments were performed with DNA synthesis in vitro.
Purified DNA polymerase a was added to the activated calf thymus DNA
in an in vitro replication system. A temperature of 46°Cfor 60 min
inhibited replication by 40%. Addition of heat-stable proteins, purified
HSP-70, bovine serum albumin, IM), or glycerol did not confer protection
to the replication system.
These studies provide new evidence that HSP-70 may confer protection
to a component of the protein synthesis machinery during thermotoler
ance.
INTRODUCTION
One of the interesting aspects of cellular response to hyperthermia is the phenomenon of thermotolerance (1-3). Cells
when exposed to a nonlethal heat shock have the ability to
acquire a transient resistance to subsequent heat challenge. The
mechanism of thermotolerance is unknown. However, this tran
sient heat resistance is coincidental with the synthesis of several
newly synthesized proteins, termed HSPs3 (4-7). Of the 3 to 4
HSPs synthesized in mammalian cells following environmental
stresses, the HSP-70 family has been the one most frequently
associated with the phenomenon of thermotolerance. Li (8)
showed a direct correlation between heat resistance and the
levels of HSP-70 and found no such correlation with other
HSPs such as HSP-89 or HSP-110.
Although several possible functional roles have been hypoth
esized for HSP-70 (9-18), the biochemical role of HSP-70
during the development of thermotolerance in mammalian cells
is unknown. HSP-70 is likely to play a structural role, since no
Received 7/19/88; accepted 12/9/88.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by Bio-Medical Research Support Grants, NIH
2S07-RR05471-25 and NIH CA 33572.
2 To whom requests for reprints should be addressed, at City of Hope National
Medical Center, Dept. of Radiation Research, 1500 E. Duarte Road, Duarte, CA
91010.
3 The abbreviations used are: HSP, heat shock protein; HSP-70, M, 70,000
heat shock protein family of proteins (others defined similarly); BSA. bovine
serum albumin; rx, reaction mixture; TCA, trichloroacetic acid.
enzymatic activity has been found to be associated with it (9).
Evidence that the HSP-70 family of proteins have binding sites
for ATP (10-12) and fatty acids (13) has led to the speculation
that HSP-70s may participate in scaffolding or dissociation of
protein complexes (14, 15). Furthermore, it has been proposed
that, during heat shock, HSP-70 binds tightly to the hydrophobic groups of other proteins and prevents them from denaturation and aggregation (15). In separate studies, Minton et al.
(19) proposed that heat-labile proteins may be stabilized nonspecifically from thermal inactivation if heated in the presence
of other heat-stable proteins. They further proposed that the
function of HSPs, which are produced in such large amounts
following heat shock, may be to nonspecifically protect other
proteins.
In the studies presented here, protective effects of HSP-70
and other agents on protein and DNA synthesis were investi
gated.
MATERIALS
AND METHODS
Cell Growth and Maintenance. Chinese hamster HA-1 cells were
grown on monolayer cultures in minimal essential medium plus 15%
fetal calf serum and 20 Mg/ml of gentamicin.
Purification of HSP-70. HSP-70 was purified according to published
procedures (12, 20). Briefly 10' Chinese hamster (HA-1) cells were
heated in a monolayer at 45°Cfor 20 min. They were then incubated
at 37°Cfor 16 h before they were trypsinized, washed with phos
phate-buffered saline and frozen at -20°C. Cells were resuspended in
hypotonie buffer [10 mM Tris-acetate (pH 7.5): 10 mM NaClrO.l mivi
EDTA], and after Dounce homogenization, the mixture was centrifuged
at 12,000 x g for 15 min. The supernatant was applied to DEAESephacell (Pharmacia), and after extensive washes with Buffer B [20
mM Tris-acetate (pH 7.5):20 mM NaChO.l mM EDTA: 15 mM 2mercaptoethanol], the proteins were eluted with the above buffer in a
linear gradient of 20 to 350 mM NaCl. The peak containing HSP-70
as determined by gel electrophoresis was then loaded on ATP-agarose
(linked through C-8) (Sigma Chemical Co.), washed with Buffer D
(Buffer B plus 3 mM MgCl2), and HSP-70 was finally eluted with Buffer
D containing 3 mM ATP (12). The purified HSP-70 was extensively
dialyzed against sterile, deionized H2O. The solution was then dried
under vacuum to powder form and dissolved in few ml of H2O. Purity
and mass of the purified HSP-70 were determined by one-dimensional
gel electrophoresis (Fig. 1). The major band in Fig. 1 shows the
inducible HSP-70, and the minor band represents the other HSP-70
family of proteins as previously reported (12, 20). The yield of the final
product was estimated by Bio-Rad protein assay (Bio-Rad) and against
increasing concentrations of bovine serum albumin run simultaneously
on one-dimensional gel electrophoresis. HA-1 cells (IO9) yielded ap
proximately 150 Mgof purified HSP-70.
In Vitro Translation System. The rabbit reticulocyte in vitro transla
tion system was purchased from Bethesda Research Laboratories,
Gaithersburg, MD. The globin mRNA was used in all studies. The rx
contained potassium acetate (2 M) (pH 7.2), 5 pCi of [35S]methionine,
25 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic
acid (pH 7.2),
40 mM KC1, 10 mM creatine phosphate, 60 fimol of each of 19 amino
acids, rabbit reticulocyte lysate, 0.2 fig of globin mRNA, and H2O. The
final volume was 30 ¡i\.At 20, 40, and 60 min at 30°Cor higher
temperatures, 7 ni of rx were removed and incubated at 30°Cfor 15
min in the presence of 1 /*!of RNase (1-mg/ml solution). This step was
done to hydrolyze radioactive amino-acyl-tRNAs. Two-^1 aliquots were
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EFFECTS OF HSP-70 ON PROTEIN AND DNA SYNTHESIS
added to the protein or DNA synthesis was chosen so as to result in
minimal inhibition of either process at 30 or 37°C,respectively.
92
RESULTS
66
Effects of Fetuin on Protein Synthesis at Elevated Tempera
tures. To investigate the temperature sensitivity of the transla
tion system, the protein synthesis was performed at 30°C(op
timum temperature), 40, and 42°C.Temperatures of 40 and
42°Cresulted in over 40 to 70% inhibition of protein synthesis
45
in all studies.
To find out whether this inhibition of globin mRNA trans
lation would be protected in the presence of nonspecific heatstable proteins, globin mRNA was translated in the presence of
various concentrations of fetuin (0.7 to 86 ^mol) at 42°Cfor
60 min (Fig. 2). Fetuin protected protein synthesis at elevated
temperatures.
Table 1 shows the cpm obtained in the above experiments.
Similar experiments were performed with ovomucoid. Ovomucoid also slightly protected the translation system at 0.30
¿/mol;however, at a lower concentration of 0.06 nmol it was no
longer protective (data not shown).
Effects of Purified HSP-70 on Protein Synthesis at Elevated
Temperatures. To examine whether the purified HSP-70 would
also be protective of mRNA translation at elevated tempera
tures, protein synthesis was performed in the presence of in
creasing concentrations (0.012, 0.025, and 0.05 /¿mol)of HSP-
31
MSFL70
Fig. 1. One-dimensional gel electrophoresis of the purified HSP-70 isolated
following 45°C/20-min heat shock treatment of Chinese hamster HA-1 cells.
Molecular weight markers shown at right.
then precipitated on glass fiber filters with 10% TCA. After several
washes in 5% TCA, the filters were dried and counted with scintillation
counting. Control groups were run simultaneously with every experi
ment. Control groups included translation at 30°Cwith or without
various supplements. The percentages shown in the figures were derived
from cpm obtained from 60-min incubation at 40 or 42"C with or
without supplements divided by cpm at 30°Cwith or without the
appropriate supplements. Background groups (-mRNA) were also run
with or without various supplements at all temperatures. A represent
ative experiment is shown in all cases; however, all experiments were
carried out at least 3 times, and statistical analysis includes the mean
±SD. For each experiment globin products were also examined in onedimensional gel electrophoresis.
Labeling and Electrophoresis. Following translation, an aliquot of
the rx was added to sodium dodecyl sulfate sample buffer (21) for onedimensional gel electrophoresis. Running gels contained 12 or 15%
acrylamide. Following electrophoresis, gels were stained with Coomassie Blue R250 in 3.5% perchloric acid, destained in 7% acetic acid,
dried, and autoradiographed on Kodak SB-5 X-ray film.
Replication System. DNA synthesis was performed as previously
described (22, 23). The replication system contained 100 n\ of the
following mixtures: 0.05 M Tris, pH 7.5; 7.5 mM MgCI2:0.5 min
dithiothreitol:0.5 mg/ml of BSA: 100 n<mo\ of each dCTP, dGTP,
dATP, and ['H]TTP (final concentration, 1000 dpm/pmol; ICN; spe
60 MIN AT 42C
Fig. 2. Effect of fetuin on the globin mRNA translation. The ability of the in
vitro translation system to direct globin synthesis from globin mRNA was meas
ured at 30, 40, or 42'C for 60 min in the presence of various concentrations of
fetuin. At the end of the 60-min incubation, (he TCA-insoluble fraction (|35S)methionine) was counted by scintillation counting.
cific activity, 47 Ci/mmol); 26 ¿igof activated calf thymus DNA (24);
and purified DNA polymerase a (22) (0.15 unit/rx; 1 unit = 1 nmol of
deoxynucleoside monophosphate incorporated in 30 min at 37°C),
which was a generous gift of Dr. Sharon Krauss, Department of
Biochemistry, University of California, Berkeley. After 60 min at 37°C
or 46°C,the reaction was stopped by the addition of 2 ml of 10% TCA
plus 20 mM sodium pyrophosphate, 0.2 ml of salmon sperm DNA (1
mg/ml), and 0.0667 ml of BSA (3 mg/ml). The precipitate was then
washed and dissolved in 0.2 M NaOH, and radioactivity was determined
by scintillation counting.
Addition of Various Agents to Translation and Replication System.
One to 5 /il of various proteins or agents were added to the reaction
mixture. The proteins, glycerol and DjO, were diluted in diethylpyrocarbonate-treated (25) sterile \\iQ (to destroy any RNase present)
before their addition into the translation system. The amount of agents
Table 1 Effects of increasing concentrations of fetuin on the globin mRNA
translation at 30 and 42'C for 60 min
Background counts without mRNA were 158,345 ±14,149 for all groups. P
< 0.001 for all comparisons with control.
±2,5
Control0.69 13°742,860
11,320338.100+
±
1.5)*(45.5
±
11,010
±1.5)
±116,250
773.205±37,590 474,525±13,050 (61.4±1.7)
488,490
±22,485
702,075±5,715
(70±3.2)
633,600+ 24,198(32.6
„¿M30'C780,400
(87±3.3)
730,516±1,28542'C254,522
' Mean ±SD.
' Numbers in parentheses, percentage of control activity.
UM3.5
UM
(iMX(>
17
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EFFECTS OF HSP-70 ON PROTEIN AND DNA SYNTHESIS
Table 2 Effects of increasing concentrations of HSP-70 on the globin mRNA translation at 30°C, 40°C, and 42°C for 60 min
Background counts without mRNA were 144,040 ±22,803 for all groups.
30'C
1,603,300 ±89,942"
Control (no HSP-70)
0.012 MM
1,499,709 ±8,507
0.025 MM
1,323,130 ±192,624
0.05 MM
844,983 ±26,550
" Mean ±SD.
* Numbers in parentheses, percentage of control activity.
' Not statistically higher than control.
d Significantly higher than control (P < 0.01).
' Significantly higher than control (P < 0.001).
40'C
884,225
914,822
1,052,425
723,729
42'C
±68,959 (55.2 ±4.3)*
±19,698(61 ±1.3)c
±29,515 (79.5 ±2.2)'
±25,980 (85.7 ±3.0)'
423,835
494,903
740,220
665,001
±4,838 (26.4 ±0.3)
±30,968 (33 ±2.0)''
±45,081 (56 ±3.4)'
±81,045 (79.0 ±9.5)'
92 _
66-
45-
31-
2160 MIN AT
40C
42°C
14-
Fig. 3. Effect of purified HSP-70 on the translation system at 40 and 42'C.
The ability of the in vitro translation system to direct globin synthesis from globin
mRNA was measured at 30, 40, or 42'C for 60 min in the presence of various
concentrations of HSP-70. At the end of the 60-min incubation, the TCAinsoluble fraction ((35S]methionine) was counted by scintillation counting.
Table 3 Globin mRNA translation at 30°C, 40°C,and 42°C in the presence and
absence of 0.025 »molof HSP-70
The background counts without mRNA were in the range of 165,810 ±11,066
for all groups.
-HSP-70
+HSP-70
30'C
20 min 620,680 ±11,250°
825,425 ±27,128
40 min 1,130,930 ±68,816
60 min l,603,300 ±89,942
1,115,395 ±42,018
1,325,130 ±192,624
40'C
20 min
40 min
60 min
454,305±68,643(73.2±11.0)* 664,440 ±18,915 (80 ±2.2)'
630,280±51,135(55.7±4.5) 821,800 ±19,857 (74 ±l.Tf
884,225±68,959(55.2±4.3) 1,052,425 ±29,515 (80 ±2.2)'
42-C
512,250 ±4,555 (62 ±0.5)'
20 min
354,245 ±24,726 (57 ±3.9)
663,220 ±26,938 (60 ±2.0)'
40 min 440,380 ±27,197 (38.9 ±2.4)
740,220 ±45,081(56 ±3.4)'
60 min 423,835 ±4,838 (26.4 ±0.3)
' Mean ±SD.
* Numbers in parentheses, percentage of control activity.
' Not significantly higher than control.
* Significantly higher than control (P < 0.01).
' Significantly higher than control (/' < 0.001).
70 (Fig. 3; Table 2). The protection of translation was evident
at both 40°Cand 42°Cfor l h and at other time points which
BG
40°C
BG
BG
Fig. 4. Autoradiographs of ["S]methionine-labeled globin after 60-min incu
bation in the presence of various concentrations of HSP-70 at control or elevated
temperatures. Globin mRNA was translated at 30, 40, and 42'C in the presence
of increasing concentrations of HSP-70 for 60 min. HG, translation with no
globin mRNA added. Molecular weights (xlO~3) are shown at the left. The minor
bands above globin (M, 12,000) are "S-binding protein and other mRNA contam
inants in the purified globin mRNA.
occurred at much lower concentrations when compared to
fetuin or ovomucoid.
Effects of BSA, Glycerol, or D2O on Protein Synthesis at
Elevated Temperatures. Since glycerol and D2O have been
shown to protect against cell kill when present during heating,
protein synthesis experiments were also performed with 2.5 /*M
BSA, 0.7 M glycerol, or 2.7 M D2O present in the reaction
mixture. BSA, glycerol, and D2O did not protect protein syn
thesis at elevated temperatures (data not shown). Higher con
centrations of these agents were inhibitory to protein synthesis
and therefore were not used.
Effects of Various Agents on DNA Synthesis at Elevated
Temperatures. The protective effects of HSP-70 and other
agents were also examined on the DNA synthesis. The optimum
temperature of DNA synthesis was at 37°C.DNA synthesis
was found to be relatively unaffected at elevated temperatures.
At 46°Cfor 60 min it was only inhibited by approximately
40%. Furthermore, the various concentrations of HSP-70,
other heat-stable proteins, such as fetuin and ovomucoid, and
agents such as BSA, glycerol, or D2O did not confer protection
to the DNA synthetic machinery (data not shown).
included 20 and 40 min of translation as it is shown in Table
3, when 0.025 /¿M
HSP-70 was added to the translation system.
Fig. 4 shows the mRNA globin translation products at 30, 40,
and 42°Cin the absence or presence of 0.025 MMHSP-70.
HSP-70 was inhibitory to the translation system at 0.05 //mol.
Higher concentrations of HSP-70 also resulted in dramatic DISCUSSION
protection of the translation system. However, such data are
In the studies presented here, we investigated the possible
not shown due to the fact that it makes the interpretation of protective effects of purified HSP-70 and other nonspecific
the results somewhat difficult. The protective effect of HSP-70 proteins on two biochemical processes in vitro. In all cases the
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EFFECTS OF HSP-70 ON PROTEIN AND DNA SYNTHESIS
concentration of the agents used was low enough to minimize
any inhibition of either process at control temperatures. The
results indicate that protein synthesis inhibition at elevated
temperatures could be protected by fetuin or ovomucoid. The
protection of translation, however, was more effective in the
presence of HSP-70 and occurred at lower concentrations. Our
results also indicate that protection of translation varied de
pending on the agent and was in the following order: HSP-70
> fetuin > ovomucoid > glycerol or D^O. Neither glycerol (23)
nor DiO (26), which has previously been shown to be protective
against heat cell kill, protected protein synthesis. Glycerol or
D2O probably protects other heat-sensitive targets in the cell.
Above results are in contrast to the work by Prenninger and Li
(Ref. 27; Footnote 4) where they observed no protection of cell
survival when RBC were loaded with fetuin or ovomucoid and
then fused with Chinese hamster HA-1 cells. However, Li et al.
(28) showed that microinjection of HSP-70 in to HA-1 cells
conferred protection against heat damage.
The ability of DNA polymerase a to replicate activated calf
thymus DNA at elevated temperatures, however, was not af
fected by any of these agents. As it has been shown previously
(Ref. 29; Footnote 5), isolated DNA polymerase a and ßare
protected by BSA and Langendorff salts when heated in the
absence of activated DNA. However, we found DNA polymer
ase a to be relatively resistant to heat when it was assayed in
the presence of activated DNA.
Protein stabilization by polyols, sugars, and small molecules
from heat denaturation is well known (30-32). The proteins
are stabilized by several factors including increased hydrophobic
interactions, hydrogen bonding, electrostatic interactions, spe
cific binding of ions and cofactors, cross-linking, and metal
complexing (30). Back et al. (30) examined the increased ther
mal stability of proteins, such as ovalbumin, lysozyme, etc., in
the presence of sucrose and glycerol. They found that both
agents strengthened the hydrophobic interaction between the
hydrophobic groups of proteins. Other studies have also dem
onstrated protein stabilization by sucrose (32). Minton et al.
(19), however, proposed that proteins may be stabilized by other
heat-stable macromolecules or proteins. Their studies suggested
that proteins such as BSA, alkaline phosphatase, and thrombin
could be protected by a variety of heat-stable proteins such as
fetuin, ovomucoid, and RNase. They found the protective ef
fects of ovomucoid > RNase > fetuin > sucrose > sorbitol. The
protection of proteins by other macromolecules from heat de
naturation resulted in the hypothesis that HSPs which are
produced in large amounts in cells following heat shock may
also protect other proteins from heat denaturation.
Since the earlier work by Minton et al. (19), there is now
accumulating evidence that HSP-70 may tightly bind to other
proteins through hydrophobic binding, thereby preventing the
protein denaturation and aggregation (15). The evidence comes
from studies which show that the addition of ATP and not the
nonhydrolyzable ATP analogues can release HSP-70 binding
from the heat-shocked nucleoli (33). As a result of such obser
vations Pelham (15) proposed a model for the function of HSP70. This model proposes that HSP-70 binds to hydrophobic
groups of a variety of proteins and prevents them from aggre
gation and denaturation. HSP-70 then is released from such
binding by using ATP hydrolysis (15). After heat shock, the
proteins can then return to their original folded state.
The conclusions derived from the studies presented here
support the earlier hypothesis put forward by others that HSP4 S. W. Prenninger and G. C. Li, personal communication.
'' I. J. Spiro i-i al., unpublished data.
70 may indeed stabilize other heat-sensitive proteins or factors
from possible aggregations or denaturation. We further propose
that one such heat-sensitive target may be the protein synthesis
machinery. Both HSP-70 and other nonspecific heat-stable
proteins may stabilize proteins, factors, polysomes, or mRNA
on the polysomes, etc., during protein synthesis at elevated
temperatures. Such heat-sensitive target(s) do not seem to be
associated with DNA synthesis.
ACKNOWLEDGMENTS
The authors wish to thank Saroj Kotecha for typing the manuscript.
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Effects of Heat Shock Proteins (Mr 70,000) on Protein and DNA
Synthesis at Elevated Temperatures in Vitro
Nahid F. Mivechi and Peter D. Ogilvie
Cancer Res 1989;49:1492-1496.
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