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RADIATION RESEARCH
137,
190-195
(1994)
Regulation
of Heat and Radiation
Stress
Responses
in
Yeast
by
hsp-104
D.
R.
Boreham and
R.
E.
J.
Mitchel
Health Sciences and Services
Division,
AECL
Research,
Chalk River
Laboratories,
Chalk
River,
Ontario KOJ
1JO Canada
Boreham,
D. R. and
Mitchel,
R. E.
J. Heat and Radiation
Stress
Response
Regulation
in
Yeast
by hsp-104.
Radiat. Res.
137,
190-195
(1994).
We have
investigated
the role
of
heat-shock
protein
104
(hsp-
104)
in
the induction
of
ionizing
radiation resistance
in
yeast.
Yeast defective in the
production
of
hsp-104
are
constitutively
sensitive to
killing by
heat
and
are
known to be
compromised
for
the induction of
thermotolerance. We confirmed that
hspl04
yeast
mutants are altered in their
ability
to become
thermotoler-
ant
by
a
heat stress.
However,
we
found that the
mutant strain
did
respond
to heat shock and
increased its resistance to a sec-
ond lethal heat
stress,
although
the
magnitude
of
the increase
was much lower than
for
the
parental
wild-type
strain. Here we
report
that the
constitutive
level of
ionizing
radiation resistance
was
higher
in
the
hspl04
mutant
compared
to
wild-type
cells and
that heat shock induced
increased radioresistance
in
both
strains.
When radiation was used as the
stressing agent,
thermal
resistance was
fully
induced in
wild-type
cells and
only
slightly
induced
in
the
hspl04
mutant.
In
comparison,
radioresistance
was
induced
in
both cell strains
by
a
radiation stress. The
increased radiation resistance was
always slightly higher
in
the
hspl04
mutant but
developed
with
similar
kinetics
in
both cell
strains.
Entry
into
stationary growth phase
is
another event
known to induce
both thermal tolerance and
radiation resistance
in
yeast.
In
these
experiments,
entry
into
stationary phase
induced
similar
levels of thermal
tolerance
in
the two strains but
higher
levels of
radioresistance
in
hspl04
mutants. The
activa-
tion of both
resistance mechanisms occurred
earlier
in
the
hspl04
mutants
compared
to
wild-type
cells. These
results show that
hsp-104
is not
essential for either the
process by
which the cell
recognizes
and
responds
to
any
of these
stresses,
or the
mecha-
nisms
by
which the cell
actually
elevates its resistance
to heat
and
radiation. We
conclude, however,
that
hsp-104
is
important
in
regulating
the
development
and
magnitude
of
induced
radia-
tion
resistance as well as
induced thermal
tolerance
in
yeast.
INTRODUCTION
Heat,
radiation or
nutrient
depletion
stress in the
yeast
Saccharomyces
cerevisiae results in
the induction
of resistance
0033-7587/94
$5.00
1
?1994
by
Radiation Research
Society.
All
rights
of
reproduction
in
any
form reserved.
to the lethal
effects of a
subsequent
challenge
by
heat
or
ion-
izing
radiation
(1).
We
have
demonstrated that induced
radioresistance results from an
increased cellular
capacity
for
recombinational-type
DNA
repair
(2),
and
that DNA
dam-
age
can act as
the
signal
for induction
(3).
The
magnitude
of
induced
thermal or radiation resistance was
shown to be
pro-
portional
to the
amount of DNA
damage (3, 4),
particularly
from
radiation-generated
hydroxyl
radicals
(5).
Our
evidence
indicates that the
nuclear
enzyme
topoisomerase
I
may
be
involved
in
down-regulating
the
responses
(6).
Stress induc-
tion
of
recombinational
repair
not
only
leads to
increased
radioresistance but also alters
the mutation
frequency
in
yeast
exposed
to a
variety
of
carcinogens
or
mutagens (7,8).
The
relationship
between
stress-induced
synthesis
of
heat-
shock
proteins
and stress-induced radiation
resistance is
under
investigation.
Recent work
has demonstrated
that the
synthesis
of
heat-shock
protein
is
regulated
by
heat-shock
transcription
factors
which,
when
activated
in
response
to
a
stress,
bind
to heat-shock elements and
regulate gene
expres-
sion
(9-11).
In
human cells
the
ability
of heat-shock
factors
to bind to
heat-shock
elements and activate
transcription
is
regulated by
heat-shock factor
phosphorylation/dephospho-
rylation
processes (11).
In
yeast,
however,
the heat-shock fac-
tor is
constitutively
bound to the
heat-shock
element
(9, 10).
Transcription
of
heat-shock
genes regulated by
transcription
factors is
controlled
by
heat-shock-dependent
phosphoryla-
tion
and activation
of the
factor,
but
binding
of
heat-shock
factors to
the heat-shock elements is
not.
Heat-shock tran-
scription
factors have
been shown to
be
responsive
to
both
heat
(10)
and
radiation stress
(11), although
it is
possible
that
different
factors
are
involved.
Recently
a
yeast
mutant
has been
described
(hspl04)
which
has a
limited
capacity
for
thermotolerance induction
by
a heat
stress
(12).
This
mutant is a
deletion
mutant
unable
to
produce
the
heat-shock
protein
of 104 kDa
molecular
weight
which is
normally
induced
in
response
to a heat
shock.
The
yeast
HSP104
gene
is
highly
conserved and is
related to the
heat-inducible
gene
HSP110 found
in
mam-
malian cells
(13).
In
mammalian
cells
hsp-110
is
present
in
90
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RADIATION
AND
hsp-104
102
-a
.E
0
0)
0)
a-
4 6
8
10
46 48 50 52
Time at
52'C
( min)
Temperature
(?C)
0 20
40
60
80
100
120
Time at 37'C
(min)
FIG.
1. Heat-induced
thermal tolerance.
Cells
in
early exponential
growth phase
were heat-shocked
at 37?C and tested for survival after a subse-
quent exposure
to
lethal
temperatures.
Panel
A:
Cells
were tested
for survival to
520C
heat.
(0)
Wild-type
cells not
heat-shocked,
(A)
hspl04
mutant
cells not
heat-shocked,
(0)
heat-shocked
wild-type
cells,
(A)
heat-shocked
hspl04
cells.
Panel B: Cells were tested
for survival
after
4
min at various
high temperatures.
Symbols
as
in
panel
A.
Panel
C: Cells were
heat-shocked
for
increasing
times and induced thermal
tolerance
was measured
by
sur-
vival after a
4-min
52?C
heating. (0)
Wild-type
cells,
(A)
hspl04
mutant cells.
non-heat-shocked
cells but
can
also be
induced
by
heat
(14).
It is a nuclear
protein
associated
with
RNA
(14);
it increases
when cells
are released
from division
delay,
and
then redis-
tributes
to the
cytoplasm
(15).
A CHO
mutant
with reduced
synthesis
of
hsp-110
(and
hsp-70)
after
heat shock
is,
like the
yeast
mutant
hspl04,
deficient
in
the
acquisition
of full ther-
motolerance
(16).
Some
evidence
suggests
that
hsp-110
may
protect
RNA
processing
and reduce
recovery delay
after a
heat
shock
(17).
In
yeast
there
appears
to be a
single
HSP104
gene,
and
as in
mammalian
cells
the
protein
is both
nuclear
and
cytoplasmic
(13, 14).
The lack
of normal
thermotolerance
induction
in
hspl04
can
be
compensated
for
by overexpression
of
hsp-70
(13).
We
have examined
the
hspl04
mutant
yeast
for
its
respon-
siveness
to
heat,
radiation
and
nutrient stress. Our
results
sug-
gest
that
hsp-104
may
be
involved
in
regulating
the
magnitude
of the resistance
induced
to radiation
as well
as to
heat.
MATERIALS
AND METHODS
Cell
strains and
culture.
The
yeast
strains used
in these
experiments
were obtained
as a
gift directly
from
Dr. S.
Lindquist
(University
of
Chicago).
The
patterns
of
protein
synthesis
of the
wild-type
and
hspl04
yeast
strains
have been
characterized
previously
by
Sanchez
and
Lindquist
(12). Haploid
Saccharomyces
cerevisiae,
both
hspl04
mutant
and
parental
wild-type
strains,
were
grown
at
21?C in
complete liquid
medium in a
shaking
incubator
as described
previously
(6).
Heat stress.
Exponentially
growing
cells
in fresh
growth
medium
were
heat-shocked
by
direct
transfer
from 21?C
to a
shaking
water
bath at
37?C.
The heat-shock
temperature
was not lethal
to either the
mutant
or
wild-type
cells.
After various
37?C
heat-shock
treatment
times,
cells
were
assessed
for the
development
of thermal
or radiation
resistance
as
described below.
Radiation
stress. Cells
were
grown
to
mid-exponential growth phase
and then cooled
to 0?C.
They
were then washed twice
in
cold 20
mM
sodium
phosphate
buffer
(pH
7.0), resuspended
in buffer
at
0?C and
irra-
diated
in the
presence
of
02.
Cells received either
100 or 250
Gy
of 60Co
y
rays
delivered
at a dose rate
of about 2
Gy/s.
These doses
were not
lethal to
the mutant
or
wild-type
cells. After
exposure,
cells were resus-
pended
in fresh medium at
the
original
cell number and incubated at
21?C.
At selected incubation
times cell
samples
were removed and
cooled to 0?C
in ice
water,
and
the
acquired
resistance was determined
as
described
later.
Nutrient
depletion
stress. Cells
in
exponential growth phase
were inoc-
ulated
into
complete
liquid
medium at 21?C. The
initial cell number
was
about
3.5
x
103
cells/ml.
Samples
were
removed at various
times for
assessment
of thermal or
radiation resistance as described
later.
As time
progressed,
cell number
increased and nutrient
supply
decreased.
Thermal resistance.
Constitutive
thermal resistance
or induced
ther-
motolerance
was assessed
by placing
1.5 ml of the cells
in medium at 0?C
into
glass
test
tubes.
The
samples
were
quickly
transferred
into a
shaking
water bath
at 52?C
or other
high
lethal
test
temperatures
for the
indicat-
ed times. At
the
end of the
exposure
to the lethal
temperature,
cells were
cooled
rapidly
in an ice bath and
plated
(at
appropriate
dilutions)
on
nutrient
agar
plates.
Cell
survival
was determined
by
scoring colony
for-
mation after
5
days
of incubation
at 21?C
(6).
Radiation
resistance.
Cells
were washed
twice and
resuspended
in 20
mM
phosphate
buffer
at 0?C.
Cell survival
was determined
after
expo-
sure
to either
a
single
dose
of 1.75
kGy
60Co
y
radiation
(bubbled
with
02)
or various
doses
to
generate
complete
survival
curves.
Cell survival
was determined
by
colony
formation
as
described
above
(6).
Data
and
figures.
All
figures
shown,
except Figs.
6 and
7,
represent
the
average
results of
three to
five
experiments.
Standard error
bars
are
shown
except
where the
error is less
than or
equal
to the
symbol
size.
RESULTS
Thermal tolerance
induced
by
heat shock.
Wild-type
and
hspl04
mutant
yeast
were
grown
at
21?C
to
early exponential
Co
0
CO
0
0)
0
a-
c
(1)
12)
CO
0
2
0.
I,
191
BOREHAM
AND MITCHEL
10-1
10-2-
ca
/
20
1
c
/
o
FIG.
2
.
.Hat-nued
radiat
rstc
Cells
\ine
arl
expngentia
1
0-4-/
10-5
I
I I
I
0
1 2
3
4
Time
at 37?C
(min)
FIG.
2. Heat-induced radiation resistance.
Cells in
early
exponential
growth phase
were heat-shocked at 37?C for
various times and
changes
in
radiation
resistance were monitored
by measuring
survival after
exposure
to a
1.75-kGy
test
dose.
(0) Wild-type
cells,
(A)
hspl04
mutant
cells.
growth phase
(5
x
105
cells/ml)
and
then
heat-shocked
at
37?C.
The resistance
of
these
cells,
and of unheated
(unin-
duced)
control
cells,
to
killing by heating
at
52?C
is shown
in
Fig.
1. The
results
show that the uninduced
hspl04
mutant
cells are
constitutively
more sensitive
to
killing
by
52?C
heat
than
the
parental wild-type
cells
(Fig. 1A),
and a similar
result was obtained at other
lethal
temperatures
(Fig.
1B).
Both
wild-type
and
hspl04
cells were
induced
by
the
37?C
heat
shock to increased levels of thermal
resistance,
but the
induced
level of
resistance
in
hspl04
cells was much lower
than
in
the wild
type
(Figs.
1A,
1B).
In
both normal
and
hspl04
control cells
(uninduced),
cell
killing
at 52?C
ceased
after 2-3
min
and
the
remaining
cells were resistant to
fur-
ther
heating (Fig. 1A).
This
phenomenon
of
induced thermal
tolerance at lethal heat
temperatures
has been
reported pre-
viously by
us
for
yeast (3,
6)
and is similar to the
response
of
mammalian cells at lower lethal heat
temperature (20).
The
results
in
Fig.
1A
show
that,
after
about 8
min
at
52?C,
the
level
of
heat
killing
in the
heat-shock-induced
hspl04
cells
was the same
as in
the uninduced
cells,
whereas
the
induced
wild-type
cells were more
resistant than the uninduced wild-
type
cells
at
all
heating
times tested.
The
data in
Fig.
1C
show
that,
as the
duration
of
the 37?C heat stress
increased,
the
hspl04
mutants
always
developed
less overall thermal resis-
tance
than
the
wild-type
cells
subjected
to
the same stress.
Heat shock and
radiation resistance.
Wild-type
and
hspl04
mutant
cells
were
compared
for their
ability
to become
resis-
tant to radiation
in
response
to a heat stress. Cells
were
grown
to
early
log phase (5
x
105
cells/ml)
at 21?C and
then
heat-shocked
in medium at 37?C
for
various
times.
Samples
were tested for
radioresistance
by measuring
survival to
a
1.75-kGy
test dose
delivered at 0?C
in the
presence
of
02.
Figure
2
shows that
both the normal and
hspl04
cells
responded
to
a
heat stress
by
increasing
their resistance
to
ionizing
radiation,
and that
in both
cases the
response
was
rapid, reaching
a maximum within
1 h
after the
beginning
of
the heat shock.
Not
only
was the initial constitutive radiore-
sistance
of
the
hspl04
mutant
slightly higher
than the wild
type
(see
also
Fig. 6),
but also the absolute
magnitude
of
the
increase
in
resistance was
greater
in
the
hspl04
mutant
cells.
Radiation-induced
thermal resistance.
Wild-type
and
hspl04
mutant
cells
were
grown
to
early
log phase
(5
x
105
cells/ml)
at 21?C. These cells received
inducing
doses of 100
(Fig.
3A)
or 250
Gy (Fig. 3B)
in the
presence
of
02
at 0?C
(100%
survival
in
both
cases)
and were
then
incubated
in
medium
at 21?C for various times. The
samples
were then
tested
for
resistance to
killing by
a
4-min 52?C heat
exposure.
The results
in
Figs.
3A
and 3B
show that radiation
exposure
induced
large
increases
in
the thermal resistance of the wild-
type
cells,
reaching
a maximum after about 4-6
h
of
incuba-
tion
at 21?C
after the radiation
exposure.
In
contrast,
the
magnitude
of the increase
in
thermal resistance induced
by
these
radiation doses in the
hspl04
mutant was much less for
all incubation times examined.
Radiation-induced
radiation resistance.
Wild-type
and
hspl04
mutant cells were
grown
to
early log phase (5
x
105
cells/ml)
at
21?C. These
cells
were
exposed
to
inducing
doses
of 100 or 250
Gy
at
0?C
in
02
and then incubated at 21?C
in
medium for various times.
Changes
in
radiation resistance
were
assessed
by measuring
survival
to a test dose of 1.75
kGy
in the
presence
of
02
at 0?C. The results
in
Figs.
4A
and
4B
show that both
wild-type
and
hspl04
mutant
cells
increased their
radiation
resistance
in
response
to radiation
stress. Radiation resistance increased with similar kinetics
in
the two cell
lines
and after 5-6
h
of
incubation
reached
simi-
lar
maximum
levels,
although
the
hspl04
mutant
always
showed
a
slightly
greater
level of radioresistance.
Figure
4C
shows
radiation
survival
curves
for
wild-type
and
hspl04
mutant cells. The
results
again
demonstrate
a
greater
response
in
the
hspl04
mutant.
Resistance induced
by
nutrient
depletion
stress. The
effects
of
a
nutrient
depletion
stress
on
both radiation and thermal resis-
tances
were
compared
in
wild-type
and
hsplO4
mutants.
Cells
were
grown
from a low cell
concentration
and
samples
taken at
various
points
in the
growth
curve,
from
very
early log phase (1
x
105
/ml)
to late
stationary phase (2
x
108/ml).
These cells
were tested for thermal
resistance
by measuring
survival after
a
4-min 52?C heat
exposure (Fig.
5)
and for
radioresistance
by
192
RADIATION AND
hsp-104
100
-
B
I
0
I
*-.
I
0
Q>
Ct%
0
c
(d
n
T
-A
I
&
0-@
)
n-4
i
I
I
I I
0
1
2
3
4
Time
at
21'C
(h)
0
1 2
3
4
Time
at 21?C
(h)
FIG.
3.
Radiation-induced
thermal resistance. Cells
in
early log phase
of
growth
were
exposed
to a
single inducing
dose
of
radiation
and then incu-
bated
at
21?C
for
various times.
Changes
in
thermal resistance
were
monitored
by
measuring
survival
after
exposure
to 52?C for
4
min.
(0) Wild-type
cells,
(A)
hsplO4
mutant
cells,
(0)
uninduced control
wild-type
cells,
(A)
uninduced
control
hsplO4
mutants. Panel A:
100-Gy y-ray inducing
dose.
Panel B:
250-Gy -y-ray inducing
dose.
measuring
survival after
a
1.75-kGy
radiation
exposure (Fig.
6).
The
hspl04
cells
were
constitutively
more sensitive to heat than
wild-type
cells
at low cell
culture
concentrations
(Fig. 5)
as
noted
previously (Fig.
1A),
but at
high
culture
density
(station-
ary phase)
both
were induced to the same level of thermal
resistance.
It is
apparent
from
Fig.
5, however,
that the
hspl04
100
100
-
10-
-
.2
CO
(U
0Q
0.
a,
(1)
.2
0
0o
c
r
0(
mutant cells
responded
earlier
to nutrient
depletion
and their
thermal resistance
began
to
increase at cell densities found at
about the
midpoint
of
the
exponential growth
phase
where no
significant
change
occurred
in
the
wild-type
cells.
When
changes
in
radiation
resistance
were monitored
(Fig.
6)
both cell strains showed somewhat similar constitu-
B
'-
II
/---
X
/
I /
1
/
0
T
I
0* ?
i
a0.
1-1
I ~~~~~~CL
,rA
0
1 2
3
4
5
6
0
1
2
3 4
5
6
0.0 0.5 1.0 1.5
2.0
Time
at
21'C
(h)
Time at 21
?C
(h)
Dose
(kGy)
FIG.
4. Radiation-induced
radiation
resistance.
Cells were
grown
to
early log phase
and
exposed
to a
100-Gy
(panel
A)
or
250-Gy
(panels
B,
C)
y-
ray
inducing
dose.
The cells were incubated
for
various
times
at 21?C and
changes
in
radiation
resistance
were monitored
by
measuring
survival
after
exposure
to
a
1.75-kGy
test dose
(panels
A,
B)
or were incubated for 5 h at
21?C
and
survival
measured
after various doses
of radiation
(panel
C).
(0)
Uninduced
control
wild-type
cells and
(A)
hspl04
mutants,
(0)
induced
wild-type
and
(A)
hspl04
mutant cells.
100
A
10o-
cr
1
cu
0
rI-.
a)
0a
1
5 6
5
6
I I I I
I I
1
-1
193
10-1-
I
5
6
10-3-
l
I
i
I I I I
BOREHAM
AND MITCHEL
102-
IA
10'
-
-
100
(D
=
10-1-
.
10-2-
0)
(0)
c,)
0)
CO
cs
a
(D
(D
I
10-
lx105
I
I I I
I
I
I
1x106
1x107
1X108
Cell
Number
(cells/ml)
x1
09
FIG.
5.
Thermal
tolerance
induced
by
nutrient
deprivation
stress. Cells
were
grown
in
complete
medium at 21?C
and
samples
were removed at
increasing
cell
concentrations. Those
cells were tested
for
survival after
exposure
to 52?C for
4
min.
(@) Wild-type
cells,
(A) hspl04
mutant cells.
tive levels of radioresistance
at
very
early log phase
(although
hsp104
mutants tended
to be
slightly
higher;
see
also
Fig.
2
at
t
=
0)
but
hsp104
mutants
reached
higher
levels of induced
radioresistance
at the
high
cell concentrations
of
stationary
phase.
The
hsp104
cells
responded
earlier
in the
growth
phase
than
the
wild-type
cells to
nutrient
depletion,
as
they
did
for thermal resistance. Radioresistance
began
to rise as
found at
about the
midpoint
of the
log-phase
cell densities
where
no
change
occurred
in
the
wild-type
cells.
DISCUSSION
Both lower
and
higher eukaryotes
respond
to a heat
stress
by
a
mechanism
known as
stress
response
and
become resistant to further
heating,
a
process
called
induced
thermotolerance.
The
actual
regulation
of this
process
is
unclear,
although
it has
been
known for some
time that the
response
(the magnitude
of the induced resis-
tance)
is
proportional
to the
severity
of
the
stress
(2-8,
19,
20).
The
resistance to
further
stress
has
usually
been
assessed
in
terms of cell
survival,
but other
more
special-
ized cellular functions have
also been shown to become
thermally
tolerant. For
example,
human natural killer cells
become
thermotolerant
in their
capacity
for
target
cell
lysis
after a
heat stress
(19). Although
there is a
strong
link
between
induced thermotolerance and the
synthesis
of
heat-shock
proteins,
these
proteins
have not
yet
been
implicated
in the
regulation
of
the
response. Using
a
yeast
10-5I I
I
I
1x105
1X106
1x107
1x108
1x
Cell
Number
(cells/ml)
109
FIG.
6. Radiation resistance induced
by
nutrient
deprivation
stress.
Cells
were
grown
in
complete
medium at 21?C
and
samples
removed as
increasing
culture
cell densities were reached. These cells
were
tested
for
survival
after
exposure
to 1.75
kGy
of
y
radiation.
(S)
Wild-type
cells,
(A)
hsp104
mutant cells.
mutant defective
in the
synthesis
of
one
of
these
proteins,
hsp-104,
we
examined
the role of this
protein
in
the stress
response
mechanism.
Yeast
respond
to a
variety
of
stresses,
including
heat,
radi-
ation
and
nutrient
depletion, by increasing
their
resistance
to
both heat and
ionizing
radiation
(1).
The
hspl04
mutant was
able to elevate its resistance to both heat
(Fig.
1)
and radia-
tion
(Fig.
2)
in
response
to a heat stress. Likewise the mutant
elevated its resistance to both heat
(Fig.
3)
and
radiation
in
response
to a radiation stress
(Fig.
4).
The
results
in
Figs.
5
and 6
show
that
nutrient
depletion
stress was also
recognized
by
the
mutant as a
signal
to elevate its resistance to both heat
and radiation.
These results
show
that
hsp-104
is
not essential
for
either the
process by
which the
cell
recognizes
and
responds
to
any
of these
stresses,
or the mechanisms
by
which
the cell
actually
elevates
its
resistance to heat and radiation.
While all of
these
stress
responses
were
qualitatively
simi-
lar,
the data show
that the
response
of the
hspl04
mutant
was
quantitatively
different
from
that
of
the
wild-type
cell.
A
defi-
ciency
in
this
protein
reduced
the extent of
thermotolerance
induced
by
heat
stress
(Fig.
1).
In
addition,
the
uninduced
hspl04
mutant was
constitutively
more sensitive to heat than
the
uninduced wild
type (Fig. 1).
When
induction of
radiore-
sistance was
examined,
however
(Fig. 2),
the
opposite
was
observed and
the
magnitude
of
the
response
in
the
hspl04
mutant was
greater
than
in
the wild
type.
The
hspl04
mutants
developed
a
markedly
lower
level of
thermal
resistance com-
100
-
10-1_
10-2-
*5
I
I I I
194
.5
RADIATION AND
hsp-104
pared
to the
wild-type
cells when
radiation was used as
the
stressing
agent
(Fig. 3).
When
radiation-induced radioresis-
tance
was
measured,
the mutant
responded
in
a
comparable
manner
(Fig.
4)
and reached
similar
levels of
radioresistance
compared
to
the
wild
type, although
the mutant
always
showed
a
slightly
greater
level
in
absolute terms.
Quantitative
differences
were also
visible
during
nutrient
depletion
stress.
The
hspl04
mutant
responded
much
earlier than the wild
type
to nutrient
depletion
and increased both its
thermal
and
radioresistance at cell number concentrations
normally
con-
sidered as
early exponential growth phase
and
where
no
changes
were
detected in
wild-type
cells
(Figs.
5 and
6).
The results
reported
here indicate
that
hsp-104
is
involved
in
the
regulation
of "stress
response"
to a
variety
of
agents.
The
mutants
which
lack
hsp-104
are
constitutively
sensitive
to heat and
develop
minimal heat-induced
thermal
tolerance,
but
these same mutants have elevated constitutive radioresis-
tance
and
develop
higher
levels
of heat-induced radioresis-
tance
compared
to
wild-type
cells.
Therefore,
like thermal
tolerance,
hsp-104
also influences
the
regulation
of radiation
resistance.
We have
previously
provided
evidence
that there
are differences
in the kinetics
of the induction of thermotol-
erance and radioresistance
(6)
even
though
common
signal-
ing pathways
are
present
(3, 20).
It is
possible
that these dif-
ferences
arise from different
mechanisms that
regulate
stress
response
and that
hsp-104 regulates
both
these
processes
but
in
different
ways.
This
hypothesis
is also
supported
by
the
observed differences
in
thermotolerance
and
radioresistance
induced
by
a radiation
stress where enhanced
radioresistance
(Fig.
4)
but reduced
thermal tolerance
(Fig. 3)
was observed.
When cells
approached
"early"
plateau growth
phase
(nutri-
ent
depletion
stress),
the
hspl04
mutant
but not
the
wild-type
cells
elevated their
resistance to
both
heat
and
radiation,
indicating
that
hsp-104
is
regulating
both
processes
in
response
to this stress.
In
conclusion,
we have demonstrated
that
hsp-104
influences
the
development
of both thermal
tol-
erance
and radiation resistance.
We conclude
that
the
data
support
the
hypothesis
that
hsp-104
is a
protein
that
regulates
the
development
and
magnitude
of induced
radiation resis-
tance as well
as induced
thermotolerance
after
cellular
stress.
ACKNOWLEDGMENTS
We
wish to thank
Dr. S.
Lindquist,
University
of
Chicago,
for her
gift
of the
yeast
strains used
in this
investigation.
We also
thank
S.
Laffre-
nier,
M.-E. Bahen and J.
Lawson for
expert
technical
assistance.
This
work was funded
by
the
CANDU
Owners
Group.
Received:
June
2,
1993;
accepted: August
4,
1993
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