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Cyclic AMP and the heat shock response in Chinese hamster ovary cells
BIOCHEMICAL
Vol. 126, No. 2, 1985
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 911-916
January 3 1, 1985
CYCLIC AMP AND THE HEAT SHOCK RESPONSE IN CHINESE HAMSTER OVARY CELLS Stuart K. Calderwood+ Mary Ann Stevenson* George M. Hahn+ *Radiology Department AO-37 Radiobiology Division Stanford University Medical Center Stanford, California 94305 Received
December
19, 1984
Heat shock leads to transient increases in CAMP levels in HA-l-CHO cells. Such pulses are correlated temporally with the induction of heat resistance (thermotolerance) and with heat shock protein synthesis. Although the kinetics of CAMP increase after heating suggest a role in thermotolerance induction, full raising CAMP levels directly using dBcAMP did not produce induced by dBcAMP may thus be thermotolerance. The resistance either a component of or different to heat-shock triggered resistance. Cells which had been made thermotolerant by heat shock did not produce a pulse in CAMP level on heating. The CAMP heat in producing system thus seemed desensitized to thermotolerant cells. 0 1985 Academic Press, Inc.
Heat
is
under
treatment
(192).
induction
of thermal
Resistance
stress from
profound
modification
translation
may
involve
regulator
(3,4)
of
transcription
few
of heat
heat
in addition
to relief
ABBREVIATIONS - CHO, Chinese hamster ovary; dBcAMP, dibutyryl cyclic AMP; HSP, heat shock
in
results
in and
"heat
shock" The
clear.
stable
CAMP, protein
(1).
observed
resistance.
are not
the
conserved,
transcription
of a soluble,
911
doses
response,
of a
response
cancer is
highly
with
and the development
by heat
heating
The response
production
of the
to
modality
a
metabolism,
to the
approach the
shock
to man (1,3).
to induction release
with
triggering
the heat
of cell
an
by non-toxic
involve
bacteria
(HSP) leading
resistance to
diverted
as
problem
reaction,
species
events
A major
appears
cellular,
proteins
investigation
They positive
of
negative
cyclic
AMP,
0006-291X/85 $1.50 Copyright 0 1985 by Academic Press, Inc. Ail rights of reproduction in any form reserved.
Vol. 126, No. 2, 1985
BIOCHEMICAL
regulation
(which
AND BIOPHYSICAL
may be mediated
RESEARCH COMMUNICATIONS
by the major
heat
shock
protein
HSP 70 (4,5)). Membranes responses
appear
of
eucaryotic
investigated it
is
cells
adenylate
a membrane
cyclic
to be intimately
EXPERIMENTAL
cyclase
induces
a good candidate
(1,6,7).
produces
multiple
as an inducer
therefore,
of thermal
a
effects;
diffusible
cellular
molecule,
events
of the heat
thermal
in the
We have,
as a mediator
enzyme which
AMP which
involved
shock
(8)
and may be
response.
PROCEDURES
We used a strain (HA-l) of CHO cells growing in monolayer. Cell culture conditions, apparatus heating and survival measurement were as described previously (6,7). Cyclic AMP was measured using the competitive binding assay of Gilman (9) on cell extracts. In brief, 2~10~ cells were lysed in 1.0 ml 4 mM EDTA and heated in a boiling water bath for 4.5 min. Denatured protein was removed by spinning at 12,000 rpm for 45 min at O'C. Similar values for CAMP concentration were obtained from cells deproteinized in acid ethanol or trichloracetic acid. Extracts were assayed for CAMP by the Gilman (9) method. Bound CAMP was separated from the free nucleotide using charcoal (10). RESULTS Heat (Fig.
caused
1).
a pulse
of cyclic
The
magnitude
independent,
but
its
dependent.
At 45'C,
decayed
to
declined heating
thermotolerance
approximately
remaining
control
of the CAMP pulses induction
reached
at
was
each
a maximum plateau
cells
temperature
were
temperature
to a maximum at 7.5 min, At
elevated
by 60 min.
to below
in HA-l
increase
and duration
by 20 min.
levels
CAMP
The duration
43'C, for
CAMP
then
reached
up to 40 min and
In each case,
prolonged
levels. corresponded
to the
temperature. value
time
(Fig.
of 2).
by 7 min at 45'C
and
30 min at 43OC.
We attempted dibutyryl
levels
to control
Resistance
occurrence
by 20 min,
reduced
the
CAMP increased
control
maximum levels
of
AMP production
to
CAMP (dBcAMP).
mimic
the heat-induced
Exposure 912
CAMP
to dBcAMP (1.0
pulse
mM) led
using to
an
Vol.
126,
No. 2, 1985
BIOCHEMICAL
AND
&a
f
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
-I-
5.0
5.0
:: -0
r
‘D 2
4.0
6 E 5 5 e 8 4 p I 0
4.0
0
2” ,o 3.c
3.0
2.0
2.0
1.0
1.0
* L
0 0
60
120
Minutes
0
180
10
0
20 Minutes
at 43%
Figure 1:Effect of Heat on CAMI Concentrations in ------CAMF levels were determined immediately after points are means of duplicate asays performed cultures. The whole experiment was repeated reproducible results.
10”
I
lo’-
/
--
I
* 1
7 .z > 5 ul
lC2-
2
/ 43Oc
45Oc
I
3
1ti43
HA-l Cells heating. Data on duplicate 4 times with
I ‘--L--~ 6
3
I
!
,i3- 1, f 12 0
40
30
at 45%
,
,
30
45
I I
15 Minutes
6
0
at 43OC
I 5
Minutes
t
1
10
15
1
2c
at 45OC
Figure 2:Kinetics of Thermotolerance Induction at 43'C and 45°C ----_Thermotoleranceasassayed as cell survival after heating for 45 min. at 45OC. The times on the abscissae refer to the durations of the initial, resistance-inducing heat treatments. Data points are means of triplicate assays. The experiment was repeated twice and gave consistent values. 913
Vol. 126, No. 2, 1985
0 .. . r:,L\ ,I\.-oI---0 I BIOCHEMICAL
2 Hours
4 in d&
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
lbl
6
0
2
4
6
Han
Figure 3:cAMP in HA-1 cells ---after ---- levels and thermal resistance incubation with dBcAMP (1.0 mM) Cells were given either a 1 hr pulse or conGo=osure to dBcAi+lF'; replication as in legend to Fig. 1. The experiment was performed 3 times and gave consistent values.
increase rise
in
cellular
in CAMP levels
Removal
levels.
analogous
to
pulse
(Fig.
by one hour
We could
that
elevations
tenfold
degree
in
rise
continuous
induced
cell
treatment of
The
than
resistance
Heating
at
thermotolerant
level
cells
(Fig.
transient
increase
base-line
on continuous
not 4).
immediately heating
rising
plateau.
in the
nucleotide
in CAMP
Both
continuous
resistance
was
[CAMP] by
levels and
(Fig.
observed
pulse
of resistance
did
tenfold
a pulse
increased
inducible
43'C
fall
shock.
survival
was not
concentration.
induce
to thermal
or 2 hr after
resistance
the maximal
by heat
A rapid,
a slowly
a rapid
thus
[CAMP] led
in
3A).
preceded
of dBcAMP at 1 hr caused
to control
A
CAMP levels
3B).
after
2
increase.
This
increasing
dBcAMP
weas considerably by heat
induce
CAMP levels
a
heating
in
thermotolerant
less
shock
(Fig.
2).
pulse
in
CAMP
neither
after
hr
nor
underwent decreased
in the
below
cells.
DISCUSSION The right
order
(Fig.
2)
time for and
scale
of CAMP pulses
the nucleotide heat
shock
induced
to be a signal protein 914
synthesis
by heating for (11)
are
of the
thermotolerance in
this
cell
Vol.
126,
BIOCHEMICAL
No. 2, 1985
AND
0
60
120
Minutes
Figure 4: Effect of Heat Thermotolerant and Control induced with a-% min/45"C Conditions were as in legend twice with similar results.
type. may
A number be
involved
heating and
leads
mediate
the
translation are
patterns for
effects
of
While by heating
is
indicate
protein
synthesis
that
a part
of the
involves
Some both
on
heat
phosphorylation shock
response; in histones
events
both
which
may
transcription
CAMP activated
result studies positive
In
pulses
and
protein
kinases
to those
induced
response
1,3),
do not
R.L.,
to heating.
have shown negative 915
of that
heat
shock
Calderwood,
S.K.
may, therefore, The full
heterogeneous
response molecular
the heat-shock regulation
do not
preliminary
induce
CAMP increase
of an amalgum
they
addition,
(Anderson,
data).
and
order (Fig.
(Fig.2).
in HA-1 cells
be merely
signals.
heat
mRNA translation,
CAMP
unpublished
the
of the
to some heat-resistance
G.M.,
be
protein
in CAMP of a similar
and Hahn,
could
that
of phosphorylation
Whether
the maximum response
studies
160
not clear.
increases lead
COMMUNICATIONS
(43'C) in --on CAMP Concentrations Thermotolerance was --HA-l Cells pretreatment 8 hr prior to heating. to Fig. 1. Experiment was repeated
indicate
factors
RESEARCH
at 43%
in the induction
(12,13,14).
involved
induce
of studies
to altered
iniitiation
BIOPHYSICAL
response
(4,5,15).
A
Vol. 126, No. 2, 1985
8lOCHEMlCAL
considerable
body of data
is
in
involved
and
thermotolerance
negative in
the rate
also
determining
Desensitization thermotolerant
cells
indicates
such
This
may
enzymes indicate (such
(161,
as HSP's)
with
cell
killing
denaturation
of
induction
might
changes
adenylate
be required
system
effecters
cyclase
to heating
at the molecular
membrane
of molecular
a
such as CAMP.
CAMP-producing
in
denaturation
of thermal
be due to heat-induced
alterations
interaction
RESEARCH COMMUNICATIONS
protein
Thus,
(1,7).
stimulus
of the
that step
of thermotolerance
to a positive
cells.
indicate
induction
regulator
addition
AND BIOPHYSICAL
structure of
in
level
in
changes
in
the
(1)
or
may
thermotolerance
or phosphodiesterase.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Plenum Hahn, G.M. (1982). Hyperthermia and Cancer. Press, New York. Overgaard, J. (ed.) 1984. Hyperthermic Oncology. Taylor and Francis, London/Philadelphia Ashburner, M. and Bonner, J.J. (1979). Cell 17:241-259. Di Domenico, B.J., Bugaisky, G.E. and Lindquist, S. (1982). Cell 31:593-603. In: Heat Shock from Bacteria to Man. Bonner, J.J. (1982). eds.) pp 147-154, Cold Spring (Schlesinger, M.J. et al, Harbor Laboratory. and Hahn, G.M. (1983). Biochim. Biophys. Calderwood, S.K. Acta 756:1-B. Fisher, G.A. and Hahn, G.M. (1982) Radiat. Res. Li, G.C.., 89:361-368. Cyclic Boynton, A.L. and Whitfield, J.F. (1983). Adv. Nucleotide. Res. 15:193-294. Gilman, A.G. (1970). Proc. Nat1 Acad. Sci. U.S.A. 67:305312. Albano, J.D.M., Ekins, R.P., Sgherzi, A.M. and Brown, B.L, Tampion, W. (1971) Biochem. J. 121:561-562. Li, G.C. and Werb, Z. (1982). Proc. Natl. Acad. Sci. USA, 79~3218-3272. Ernst, V., Baum, E.Z. and Reddy, P. (1982). In: Heat Shock from Bacteria to Man. Schlesinger, M.J. zal, eds) pp 215-225. Cold Spring Harbor Laboratory. Glover, C.V.C. (1982). Proc. Natl. Acad. Sci. USA 79:17811785. Sanders, M.A., Feeney-Triemer D., Olsen, A.S. and FarrellTowt, J. (1982). In:. Heat Shock from Bacteria to Man. pp 235-242, Cold Spring Harbor Laboratory. Forces, V., Pellicer, A., Axel, R. and Meselson, M. (1981) Proc. Natl. Acad. Sci. Usa 78:7038-7042. Rodbell, M. (1980) Nature 284:17-21.
916
Vol. 126, No. 2, 1985
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 911-916
January 3 1, 1985
CYCLIC AMP AND THE HEAT SHOCK RESPONSE IN CHINESE HAMSTER OVARY CELLS Stuart K. Calderwood+ Mary Ann Stevenson* George M. Hahn+ *Radiology Department AO-37 Radiobiology Division Stanford University Medical Center Stanford, California 94305 Received
December
19, 1984
Heat shock leads to transient increases in CAMP levels in HA-l-CHO cells. Such pulses are correlated temporally with the induction of heat resistance (thermotolerance) and with heat shock protein synthesis. Although the kinetics of CAMP increase after heating suggest a role in thermotolerance induction, full raising CAMP levels directly using dBcAMP did not produce induced by dBcAMP may thus be thermotolerance. The resistance either a component of or different to heat-shock triggered resistance. Cells which had been made thermotolerant by heat shock did not produce a pulse in CAMP level on heating. The CAMP heat in producing system thus seemed desensitized to thermotolerant cells. 0 1985 Academic Press, Inc.
Heat
is
under
treatment
(192).
induction
of thermal
Resistance
stress from
profound
modification
translation
may
involve
regulator
(3,4)
of
transcription
few
of heat
heat
in addition
to relief
ABBREVIATIONS - CHO, Chinese hamster ovary; dBcAMP, dibutyryl cyclic AMP; HSP, heat shock
in
results
in and
"heat
shock" The
clear.
stable
CAMP, protein
(1).
observed
resistance.
are not
the
conserved,
transcription
of a soluble,
911
doses
response,
of a
response
cancer is
highly
with
and the development
by heat
heating
The response
production
of the
to
modality
a
metabolism,
to the
approach the
shock
to man (1,3).
to induction release
with
triggering
the heat
of cell
an
by non-toxic
involve
bacteria
(HSP) leading
resistance to
diverted
as
problem
reaction,
species
events
A major
appears
cellular,
proteins
investigation
They positive
of
negative
cyclic
AMP,
0006-291X/85 $1.50 Copyright 0 1985 by Academic Press, Inc. Ail rights of reproduction in any form reserved.
Vol. 126, No. 2, 1985
BIOCHEMICAL
regulation
(which
AND BIOPHYSICAL
may be mediated
RESEARCH COMMUNICATIONS
by the major
heat
shock
protein
HSP 70 (4,5)). Membranes responses
appear
of
eucaryotic
investigated it
is
cells
adenylate
a membrane
cyclic
to be intimately
EXPERIMENTAL
cyclase
induces
a good candidate
(1,6,7).
produces
multiple
as an inducer
therefore,
of thermal
a
effects;
diffusible
cellular
molecule,
events
of the heat
thermal
in the
We have,
as a mediator
enzyme which
AMP which
involved
shock
(8)
and may be
response.
PROCEDURES
We used a strain (HA-l) of CHO cells growing in monolayer. Cell culture conditions, apparatus heating and survival measurement were as described previously (6,7). Cyclic AMP was measured using the competitive binding assay of Gilman (9) on cell extracts. In brief, 2~10~ cells were lysed in 1.0 ml 4 mM EDTA and heated in a boiling water bath for 4.5 min. Denatured protein was removed by spinning at 12,000 rpm for 45 min at O'C. Similar values for CAMP concentration were obtained from cells deproteinized in acid ethanol or trichloracetic acid. Extracts were assayed for CAMP by the Gilman (9) method. Bound CAMP was separated from the free nucleotide using charcoal (10). RESULTS Heat (Fig.
caused
1).
a pulse
of cyclic
The
magnitude
independent,
but
its
dependent.
At 45'C,
decayed
to
declined heating
thermotolerance
approximately
remaining
control
of the CAMP pulses induction
reached
at
was
each
a maximum plateau
cells
temperature
were
temperature
to a maximum at 7.5 min, At
elevated
by 60 min.
to below
in HA-l
increase
and duration
by 20 min.
levels
CAMP
The duration
43'C, for
CAMP
then
reached
up to 40 min and
In each case,
prolonged
levels. corresponded
to the
temperature. value
time
(Fig.
of 2).
by 7 min at 45'C
and
30 min at 43OC.
We attempted dibutyryl
levels
to control
Resistance
occurrence
by 20 min,
reduced
the
CAMP increased
control
maximum levels
of
AMP production
to
CAMP (dBcAMP).
mimic
the heat-induced
Exposure 912
CAMP
to dBcAMP (1.0
pulse
mM) led
using to
an
Vol.
126,
No. 2, 1985
BIOCHEMICAL
AND
&a
f
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
-I-
5.0
5.0
:: -0
r
‘D 2
4.0
6 E 5 5 e 8 4 p I 0
4.0
0
2” ,o 3.c
3.0
2.0
2.0
1.0
1.0
* L
0 0
60
120
Minutes
0
180
10
0
20 Minutes
at 43%
Figure 1:Effect of Heat on CAMI Concentrations in ------CAMF levels were determined immediately after points are means of duplicate asays performed cultures. The whole experiment was repeated reproducible results.
10”
I
lo’-
/
--
I
* 1
7 .z > 5 ul
lC2-
2
/ 43Oc
45Oc
I
3
1ti43
HA-l Cells heating. Data on duplicate 4 times with
I ‘--L--~ 6
3
I
!
,i3- 1, f 12 0
40
30
at 45%
,
,
30
45
I I
15 Minutes
6
0
at 43OC
I 5
Minutes
t
1
10
15
1
2c
at 45OC
Figure 2:Kinetics of Thermotolerance Induction at 43'C and 45°C ----_Thermotoleranceasassayed as cell survival after heating for 45 min. at 45OC. The times on the abscissae refer to the durations of the initial, resistance-inducing heat treatments. Data points are means of triplicate assays. The experiment was repeated twice and gave consistent values. 913
Vol. 126, No. 2, 1985
0 .. . r:,L\ ,I\.-oI---0 I BIOCHEMICAL
2 Hours
4 in d&
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
lbl
6
0
2
4
6
Han
Figure 3:cAMP in HA-1 cells ---after ---- levels and thermal resistance incubation with dBcAMP (1.0 mM) Cells were given either a 1 hr pulse or conGo=osure to dBcAi+lF'; replication as in legend to Fig. 1. The experiment was performed 3 times and gave consistent values.
increase rise
in
cellular
in CAMP levels
Removal
levels.
analogous
to
pulse
(Fig.
by one hour
We could
that
elevations
tenfold
degree
in
rise
continuous
induced
cell
treatment of
The
than
resistance
Heating
at
thermotolerant
level
cells
(Fig.
transient
increase
base-line
on continuous
not 4).
immediately heating
rising
plateau.
in the
nucleotide
in CAMP
Both
continuous
resistance
was
[CAMP] by
levels and
(Fig.
observed
pulse
of resistance
did
tenfold
a pulse
increased
inducible
43'C
fall
shock.
survival
was not
concentration.
induce
to thermal
or 2 hr after
resistance
the maximal
by heat
A rapid,
a slowly
a rapid
thus
[CAMP] led
in
3A).
preceded
of dBcAMP at 1 hr caused
to control
A
CAMP levels
3B).
after
2
increase.
This
increasing
dBcAMP
weas considerably by heat
induce
CAMP levels
a
heating
in
thermotolerant
less
shock
(Fig.
2).
pulse
in
CAMP
neither
after
hr
nor
underwent decreased
in the
below
cells.
DISCUSSION The right
order
(Fig.
2)
time for and
scale
of CAMP pulses
the nucleotide heat
shock
induced
to be a signal protein 914
synthesis
by heating for (11)
are
of the
thermotolerance in
this
cell
Vol.
126,
BIOCHEMICAL
No. 2, 1985
AND
0
60
120
Minutes
Figure 4: Effect of Heat Thermotolerant and Control induced with a-% min/45"C Conditions were as in legend twice with similar results.
type. may
A number be
involved
heating and
leads
mediate
the
translation are
patterns for
effects
of
While by heating
is
indicate
protein
synthesis
that
a part
of the
involves
Some both
on
heat
phosphorylation shock
response; in histones
events
both
which
may
transcription
CAMP activated
result studies positive
In
pulses
and
protein
kinases
to those
induced
response
1,3),
do not
R.L.,
to heating.
have shown negative 915
of that
heat
shock
Calderwood,
S.K.
may, therefore, The full
heterogeneous
response molecular
the heat-shock regulation
do not
preliminary
induce
CAMP increase
of an amalgum
they
addition,
(Anderson,
data).
and
order (Fig.
(Fig.2).
in HA-1 cells
be merely
signals.
heat
mRNA translation,
CAMP
unpublished
the
of the
to some heat-resistance
G.M.,
be
protein
in CAMP of a similar
and Hahn,
could
that
of phosphorylation
Whether
the maximum response
studies
160
not clear.
increases lead
COMMUNICATIONS
(43'C) in --on CAMP Concentrations Thermotolerance was --HA-l Cells pretreatment 8 hr prior to heating. to Fig. 1. Experiment was repeated
indicate
factors
RESEARCH
at 43%
in the induction
(12,13,14).
involved
induce
of studies
to altered
iniitiation
BIOPHYSICAL
response
(4,5,15).
A
Vol. 126, No. 2, 1985
8lOCHEMlCAL
considerable
body of data
is
in
involved
and
thermotolerance
negative in
the rate
also
determining
Desensitization thermotolerant
cells
indicates
such
This
may
enzymes indicate (such
(161,
as HSP's)
with
cell
killing
denaturation
of
induction
might
changes
adenylate
be required
system
effecters
cyclase
to heating
at the molecular
membrane
of molecular
a
such as CAMP.
CAMP-producing
in
denaturation
of thermal
be due to heat-induced
alterations
interaction
RESEARCH COMMUNICATIONS
protein
Thus,
(1,7).
stimulus
of the
that step
of thermotolerance
to a positive
cells.
indicate
induction
regulator
addition
AND BIOPHYSICAL
structure of
in
level
in
changes
in
the
(1)
or
may
thermotolerance
or phosphodiesterase.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
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