Vol. 2, 1649-1657,
Advances
October
p53
Agents
Protein
in Human
Yang,
and
James
Sanford
R.
Potentiates
Colon
Eshleman,
Cytotoxicity
Cancer
Nathan
A.
DNA
Berger,
Ireland
D. Markowitz2
Cancer
Center,
University
Hospitals
of Cleveland
via
this
Gl
DNA
(13-15),
Abstract
p53
is induced
by DNA
damage.
DNA
cell
death via apoptosis.
Wild-type
p53 in different
cell types has
similarly
been
associated
with either
enhancement
of or
increased
resistance
to the cytotoxicity
apeutic
agents.
We have
line bearing,
in addition
an exogenous
wild-type
control
p53
allele
of the lac repressor.
that
but
However,
induction
erfully
that
potentiates
the
cancer
the
of wild-type
of both
5-fluorouracil,
two agents
that are used
clinically
in the
treatment
of colorectal
cancer.
We also find that induction
of wild-type
member
p53
of the
potentiates
camptothecin
clinical
activity
against
that the common
loss
cancers
may
play
tumors
to anticancer
may not be directly
suggest
that
wild-type
the cytotoxicity
of drugs
that
a
also
has
in the
clinical
resistance
agents.
Although
some
killed by p53 gene therapy,
alteration
increase
of some
their
these
formed
colon
adopted
this
presence
of
in
have
adopted
gene
Introduction
altered
genetic
combination
70%
nuclear
locus
of
of human
deletion
colorectal
phosphoprotein
gene
in human
and/or
cancers
(3).
increases
the
most
( I , 2) and
mutation,
tumors
that
is
specifically
The
p.53
in level
gene
commonly
is,
by
lost
encodes
in response
the
a
in
a
to
7/23/96;
accepted
8/15/96.
by PHS awards
ROl CA 57208,
P01 CA
S 11 83, ACS Grant FRA-45 1 , and by P3 CA 4370301
(to S. D. M.) to
the Case Western
Reserve
University
Cancer
Center
and by grants
from
the Ohio Cancer
Research
Associations
(to S. D. M.) and from the ACS,
Cuyahoga
County
Unit (to J. R. E., S. D. M.).
2 To whom requests for reprints
should
be addressed,
at UCRC#2,
Room
200, Ireland
Cancer
Center,
1 1001 Cedar Road, Cleveland,
OH 44106.
Phone: (216) 844-8237;
Fax: (216) 844-8230.
likely
line with
has
eliminated.
We
endogenous
on
growth
not induce
wild-type
transformed
p53
(26).
such
the
en-
inducible
experimental
mutant
colon
Stable
alleles
in
and
mutant
upon
have
arrest
expression
Gl
(27).
are: 5-FU,
from
the
p53
allele
SW480
under
In the basal
state,
of IPTG,
of the
cycle,
not,
even
by
it strongly
5-fluorouracil;
cell
after
show
itself,
several
that
trigger
enhances
IPTG,
the
the
that
phase
now
cell
the wild-
previously
induces
We
in
we
in 5W480
death
cells,
of
allele
(28).
line
Accordingly,
addition
or cell
does
epithelial
cell
in tumors
demonstrated
p53
in the
cancer
reintroduction
possible
by deriving
wild-type
3 The abbreviations
used
thiogalactopyranoside.
of
which
allows
and
been
are
We
reexpression
colon
effects
in
p53
the 5W480
not
studies
apoptosis
p53
the
restoring
is silent,
reversible
for successful
cancers
addition,
mutant
alleles
is expressed.
this
been
strategies
mutant
as a model
generally
of
the
lines.
has
expression
of a
in which
with
of the wild-type
are
allele
have
compared
wild-type
allele
We
the functions
is tolerated
into
In
on
in trans-
is mutant.
in tumors
line bearing
a wild-type
of the lac repressor
(28).
type
precell
effects
p53
that
depend
p53
alleles
our
p53
because
mutant.
cell
performed
and
to two
used
wild-type
the
wild-type
allele
would
of the effects
of wild-type
revised
supported
allele
wild-type
are
p53
wild-type
on inactivating
in which
also
used
that
supported
17-24)
to examine
be different
of wild-type
p53
relied
it is possible
course
therapy
(10,
of both
line a daughter
inducible
control
does
Received
5/17/96;
I This
work
was
the
this
p53 in a cell
which
have
suppressor
cells
will
isogenic
which
tumor
which
been
the role
of restoring
wild-type
alleles
wild-type
p53
the
have
systems
chosen
because
allele
We have
in
have
have
epithelial
p53
effects
poten-
in clinical
practice
(25). Many
role of p53 in modulating
damage
course
wild-type
otherwise
therapy.
The
we
to DNA
could
by agents
the
damage
contrast,
tumors
line
p53
to killing
of
Alterna-
resistance
of colorectal
cancers
and radiation,
that are commonly
examining
comparison
gene
cell
examines
DNA
damage.
wild-type
of these
malignancies
to DNA
In
wild-type
to induce
Both
study
studies
dogenous
of these
to cytotoxic
present
reintroduction
cancer
cells
our findings
cancers
sensitivity
damage.
cells
enhancing
the resistance
DNA
of cells
p53
transformed
transformed
By
enhance
apoptosis,
and
to facilitate
some
virally
induce
sensitivity
mutant p53 in modulating
therapeutic
agents,
5-FU3
p53
colon cancer.
These findings
suggest
of wild-type
p53 in many
colorectal
a role
genetic
p53 may
family
of topotecan,
that
7, 8),
of wild-type
(16).
potentially
agents
induces
(5,
suggested
in
in some
p53
cycle
restoration
tumors
p53 could
by
elevated
cell
apoptosis
solid
in different
The
types,
of the
p53 has been
(10-12),
the
findings
responses
and
cells
DNA
p53.
pow-
irradiation
in some
induce
cell
phase
induce
potentiating
responses
p53
p53
and
by
Gl
In contrast,
directly
increase
by
9).
(5,
can
tially
vious
the regulat-
of wild-type
wild-type
killing
to treat
in these cells induces
not induce
cell death.
does
cytotoxicity
ther-
cancer
cell
p5.3 alleles,
is under
Induction
by isopropyl-I-thiogalactopyranoside
a reversible
growth
arrest
we find
of many
constructed
a colorectal
to endogenous
mutant
to
tively,
In different
cell types, this induction
is suggested
either to facilitate
repair
by inducing
a cell cycle pause
or to potentiate
arrest,
wild-type
cells
some
in the
hematopoietic
Ohio 44106
In
arrest
repair
function
[B. Y.,
(4-6).
growth
repair,
Wild-type
of Therapeutic
damage
cell
J. R. E., N. A. B., S. D. M.], Cleveland,
able
1649
Cells1
Department
of Molecular
Biology and Microbiology
[B. Y.,
S. D. M.], Department
of Medicine
[B. Y., N. A. B., S. D. Ml, and
Department
of Pathology,
Case Western Reserve University
[J. R. E.],
and
Research
in Brief
Wild-Type
Bin
Clinical Cancer
1996
a
and
days
although
death
the
isopropyI--
Downloaded from clincancerres.aacrjournals.org on July 12, 2017. © 1996 American Association for Cancer
Research.
of
sen-
1650
Wild-Type
p53
sitivity
Potentiates
of these
Cytotoxicity
cells
to killing
by both
radiation
and
chemo-
Hour
24
6
12
24
48
72
-
+
+
+
+
+
therapy.
IPTG
Materials
and
Methods
Cell Lines
lon
cancer
GH2,
and Culture.
cell
bearing
line
(ATCC
an empty
an IPTG-inducible
control
expressing
plasmid
the
p53
under
(WTd),
or no eDNA
insert
eDNA
repressor
(GH2;
Ref.
ible
wild-type
p53,
were
to cytotoxic
DMEM
with
10%
a humidified
in 100-mm
tissue
time 0, the cells
was
delivered
and
400
-y-irradiator
at a rate
Topotecan
ecin;
PA)
-50
was
Kline
time point.
commenced
exposure
Assay.
cells
time
point
Mark
For all
were
seeded
at doses
I model
in the
of 25,
68
presence
Pharmaceuticals,
as 1-h exposure
‘37Cs
of
IPTG.
also
King
colonies
were
ethanol.
Surviving
periment
were
damaging
fixed
and
agent.
stained
colonies
at the 24-h
> l000/mmol)
was
and
buffer
ex-
to methionine-free
growing
DMEM
for
L-[355]methionine
2 h. The
(PBSTD)
cells
containing
I % Triton
collected.
The
were
SDS,
for
10 mm,
incorporated
with
G and
p.g/ml
(20
overnight.
PAGE
and
A (Oncogene
p.1) pAbl62O
The
were
in cold
visualized
ing.
One
trophoresis
washed
PBSTD
hundred
on
Science,
Approximately
with
50
lysis
were
NY)
and
Science)
resolved
three
buffer
times
with
cold
(pH
0.5
(Sigma)
bated
2
at 4#{176}C
for
1 h.
precipitated
agarose
X
the
106
cells
peroxidase-conjuwere visualized
Arlington
incubated
was
added,
was
by ethanol
gel,
and
were
and
and
visualized
after
was
containing
10 mi
mixture
sodium
with
acetate,
by ethidium
EDTA,
Poole,
Mannheim,
I h at 50#{176}C.Then,
the
by
(29).
(Sigma,
K (Boehringer
and
treat-
isolated
Cidlowski
N-lauroyl-sarkosine
extracted
elec-
fragmenta-
harvested
of Compton
for
by
DNA
DNA
proteinase
and
determined
of
or both.
in a solution
0.5%
DNA
was
presence
or 5-FU,
lysed
8.0),
p.g/ml
IN),
RNase
1
IPTG
were
Tris
and
of
of the method
dianapohis,
was
In-
0.5
further
p.g/ml
incu-
phenol/chloroform,
analyzed
bromide
on a 2%
staining.
by 8% SDSResults
by autoradiography.
and Western
incubated
with
either
pellets
mM
UK),
was
Keene,
(Oncogene
(Amersham,
Apoptosis
determination
a modification
Cell
of horseradish
reactive
proteins
of Apoptosis.
ment
soluble
Schuell,
to WAF1
chemiluminescence
tion.
sodium
&
specific
IL).
then
1 ml
(Schleicher
antibodies
application
antibodies,
enhanced
Heights,
and
Uniondale,
Oncogene
(Ab-5;
immunoprecipitates
Protein
Extraction
nentially
growing
cells
points
Science).
After
gated
secondary
0.5%
radioactivity
with
trophoretic
activity
lysed
0. 1%
X-lOO
2 h and
(specific
membrane
blotted
cells
measured
by scintillation
counting
after
trichloroacetic
acid
precipitation.
Equal counts from different
labeled
protein
lysates
were incubated
with 20-pA agarose
beads
conjugated
to staph
proteins
and
with
in 50%
of each
nitrocellulose
p.M
NH),
Assay
100 p.Ci/ml
and
were
surviving
blue
dishes
Exponentially
at 37#{176}Cfor
deoxycholate,
2 weeks,
methylene
triplicate
the times shown.
Immunoprecipitates
were resolved
by 8% SDS-PAGE.
B, induction
of WAF1/CIP1
protein
assayed
by Western
analysis
of
duplicate
cell lysates.
Western
analysis
for actin was performed
on the
same filter to verify
equal loading
of cell protein
from each sample.
C,
assay of DNA fragmentation
by electrophoresis
on 2% agarose
gels of
DNA prepared
from duplicate
cell lysates.
counted.
transferred
with
with
from
Immunoprecipitation.
incubated
After
1 IPTG treatment
of WTd8
cells. A, induction
of wild-type
p53
detected by immunoprecipitation
with wild-type
p53-specific
pAbl62O
of lysates of cells grown in the absence ( - ) or presence ( + ) of IPTG for
Fig.
of Prussia,
commencing
was maintained
continuously
to 24 h. Thus, in all
IPTG treatment
was continued
for 24 h after
to the DNA
extracts
in
at 37#{176}C
in
5-FU (Smith
Kline Beecham
Pharmaceuticals)
at the zero time point concurrently
with IPTG,
5-FU exposure
the experiments,
of lysis
24-h
cGy/min
Beecham
delivered
C
of sensicultured
(l0-hydroxyl-9-dimethylaminomethyl-(S)-camptoth-
Smith
were
Formation
in a Shepherd
of
Actin
-
of
12 h before IPTG treatment.
At
with 5 ms IPTG
for 48 h.
at the
cGy
either
MT2),
serum
growing
culture
dishes
were incubated
WAF1
CO2.
exponentially
100,
200,
studies
5%
-
WTd8
and
of the induc-
were
lines
B
a second
homogeneity
calf bovine
with
Radiation
operator,
the
and Colony
experiments,
lac
Two subclones,
tight regulation
heat-inactivated
Treatment
with
143 Val-Arg;
cell
an
were
constitu-
along
for subsequent
All
atmosphere
Drug
of these
used
agents.
bearing
cell lines
To maintain
p53
p
are:
bears
a plasmid
of the
the WTd cell line, it was recloned.
WTdl9,
which
both demonstrate
tivity
with
(codon
28).
MT2,
A
co-
study
which
These
protein,
p53
SW480
in this
vector;
(28).
control
mutant
the
and WTd,
of SW480
lac
containing,
wild-type
used
expression
p53
by transfection
of
228)
p53 eDNA;
wild-type
constructed
tively
CCL
mutant
IPTG-inducible
Derivatives
Blot Analysis.
IPTG
at selected
PBS.
Cells
for
10 mm
and
collected
mg of protein
12.5%
Laemmli
lysate
gradient
were separated
gel, transferred
Expotime
Wild-Type
lysed
Cells.
WTd,
by scrap-
growth
were
by electo 0.2
p53
Does
Not
Induce
Apoptosis
We reported
previously
that
in the SW480
induction
of wild-type
p53 by IPTG induces
arrest
in the
Gl
we used two subclones,
rived from WTd cells
phase
ofthe
WTd8
and that
cell
cycle
(28).
in
WTd
daughter,
a reversible
In this
study,
and WTd19,
that are both deretain the same inducible
regu-
Downloaded from clincancerres.aacrjournals.org on July 12, 2017. © 1996 American Association for Cancer
Research.
Clinical
Cancer
1651
Research
M12
GH2
1000
IPTG
-0--
100
-
IPTG+
0
100
0
0
U.
0
C
10
3
10
Cl)
Fig.
2
Potentiation
diation
genie
shown
livered
the
of cytotoxicity
by wild-type
of -y-irra-
p53 expression.
Clono-
survival
of colorectal
cancer
cells
is
at different
doses
of -y-irradiation
dein the presence
(#{149})
or absence
(0) of
inducing
SW480
control
inducible
agent
IPTG.
Derivatives
treated
are: GH2,
bearing
expression
vector;
MT2,
mutant
p53 eDNA;
and
WTd19,
both clones
0
100
200
300
0
400
100
200
300
400
300
400
of
WTd19
WTd8
an empty
bearing
an
WTd8
and
1000
1000
of WTd cells that bear an
inducible
wild-type
p53 eDNA.
shows
means
of colony
numbers
triplicate
experiments;
bars, SE.
The figure
obtained
in
100
100
10
10
C
0
C.)
0
U.
0
C
3
Cl)
.1
.1
0
100
200
300
400
0
100
200
cGy
lation
of p53
these
cells
detected
which
1A).
expression
with
by
increased
reaching
mutant
WTd
with
the
Treatment
antibody
wild-type
p53
Western
pAb 1620,
protein
protein
as early
as 6 h, and gradually
6 to 72
h after
addition
of IPTG
parental
WTd
cell
line,
repressed
(Fig.
in
1A),
of the endogenous
induction
of wild-type
induced
expression
blot
was
constant
(Fig.
induced
to that
was
p53
p53
was
equal
protein
of
wild-type
p53
a level approximately
p53 protein
(28).
As in the
of
of wild-type
of IPTG,
from
cells.
expression
recognizes
expression
the absence
original
induced
immunoprecipitation
specifically
The
as the
IPTG
cGy
of
analysis
evident
between
of WAF1/CIP1
by
24 and
protein
of p53 protein
(compare
demonstrate
that IPTG
MT2
cells
GH2
cells
that
that
Examination
contain
contain
WAFI/CIP1
protein
detectable
by
12 h of IPTG
1B, WAF1/CIP1
treatment
and remained
72 h of IPTG
treatment.
(28).
As
lagged
shown
-6
in
Fig.
h behind
The
induction
the IPTG
induction
Fig. 1, A and B). Control
did not induce
WAF1/CIPI
experiments
in either
an inducible
vector
an empty
by electrophoresis
mutant
vector
control
of genomic
p53
plasmid
DNA
or in
(28).
extracted
Downloaded from clincancerres.aacrjournals.org on July 12, 2017. © 1996 American Association for Cancer
Research.
1652 Wild-Type
p53 Potentiates
Cytotoxicity
Table
Dose enhancement
enhancement
of each agent
factor
tested.
factor
is the
1
Wild-type
p53
potentiation
is the ratio of the dose required
ratio
of the
percentage
of cell
Dose
Agent
Radiation
5-FU
Topotecan
of WTd
to achieve
survival
in the
enhancement
killing
by therapeutic
agents
LD90 in the absence
absence
versus
versus
the presence
of wild-type
the presence
of wild-type
p53 calculated
factor
Cytotoxicity
enhancement
p53. Cytotoxicity
at the highest
dose
factor
WTd8
WTd19
WTd8
WTdl9
1.8
4.0
2.1
1.5
3.8
2.9
40
38
7
10
24
4
MT2
GH2
1000
1000
IPTG
-0--
.
g
IPTG
-0--
-
IPTG+
-
IPTG#{247}
0
100
0
U.
C
10
10
Cl)
Fig.
.
I
I
I
0
5
10
I
I
0
20
#{149}I
#{149}I
#{149}I
5
10
20
wild-type
1000’
IPTG
-0-
.
100’
100’
10
10’
Potentiation
of cytotoxicity
p53 expression.
Clonogenic
of colorectal
WTd1 9
WTd8
1000
3
-
IPTG+
cancer
cells is shown
of 5-FU by
survival
at different
doses of 5-FU delivered
in the presence
(#{149})
or
absence
(0) of the inducing
agent
IPTG.
Derivatives
of SW480
treated
are: GH2,
bearing
an empty control
expression
vector;
MT2, bearing an inducible
mutant
p53 eDNA;
and WTd8
and WTdI9,
both clones
ofWTd
cells that bear
an inducible
wild-type
p53 eDNA.
The figure
shows
means
of colony
numbers
obtained
in
triplicate
experiments;
bars, SE.
0
0
0
IU.
0
C
Cl)
1’
.1
.1
0
5
5-Fluorouracil
10
20
(pM)
.
0
5
5-Fluorouracil
10
20
(pM)
Downloaded from clincancerres.aacrjournals.org on July 12, 2017. © 1996 American Association for Cancer
Research.
Clinical
Cancer
Research
10
100
1
10
100
Topotecan
(jiM)
GH2
MT2
C
0
0
0
aU.
0
C
>
3
Cl)
Fig. 4 Potentiation
of cytotoxicity
of topotecan
by wild-type
p53 expression.
Clonogenie
survival
of colorectal
cancer
cells is
shown
at different
doses of topotecan
delivered in the presence
(#{149})
or absence (0) of
the inducing
agent
IPTG.
Derivatives
of
SW480
treated
are: GH2, bearing
an empty
control
expression
vector;
MT2, bearing
an
inducible
mutant
p53 eDNA;
and WTd8
and
WTd19,
both clones
0
.1
1
WTd1 9
WTd8
of WTd cells that bear
an inducible
wild-type
p53 eDNA.
The figure shows
means
of colony
numbers
obtamed
in triplicate
experiments;
bars, SE.
C
0
0
0
U.
0
C
3
Cl)
0
.1
1
Topotecan
from
the above
cells
of WAF1/CIP1
showed
did not
that induction
activate
fragmentation
was detected
induction
of wild-type
p53
The
p53
Wild-Type
human
colon
allele
arrest
has
(6,
an
24).
effect
clonogenic
ence
and
or
To
assay
absence
no
determine
of
whether
the
in
cytotoxicity
induction
(Fig.
SW480,
of
of
after
daughter
DNA
loss
of
we
p53
WTd
dramatically
72 h of
Cells
to Irradiation.
contains
only a mutant
Gl
Table
wild-type
in
in
and
p53
the
by
the
pres-
SW480
0
cells.
.1
the
-10-fold
the
vector
(Fig.
alone
Wild-Type
5-FU,
chemotherapeutic
cells
radiosensitivity
an inducible
2).
p53
Sensitizes
a thymidylate
agent
induction
in WTd
(Fig.
(Fig.
of wild-type
---40-fold
of
MT2
mutant
WTd
used
or
p53
cells
IPTG
has no
Cells
inhibitor,
for
GH2
allele
the
cells
2 and
p53 in-
in WTd8
2). In contrast,
synthetase
currently
p53
to -irradiation
of ‘y-irradiation
in WTd19
on
of wild-type
sensitivity
dose,
cytotoxicity
bear
though
the
1). At a 400-cGy
respectively,
a
Expression
enhances
influence
checkpoint
compared
100
(pM)
creases
-y-irradiation
wild-type
p53 and
1C). No
radiation-induced
radiosensitivity
the
of wild-type
in WTd8
cells even
by IPTG addition.
p53 Sensitizes
WTd
cancer cell line SW480
exhibits
on
apoptosis
10
cells
that,
or an empty
to
5-FU.
is
treatment
Al-
the
main
of
Downloaded from clincancerres.aacrjournals.org on July 12, 2017. © 1996 American Association for Cancer
Research.
co-
1653
1654
p53 Potentiates
Wild-Type
Cytotoxicity
lon cancer patients,
less than one-third
of colon cancers
to such therapy
(25). To determine
whether
induction
type
p53
would
cancer
cells,
in the
presence
enhance
the
we analyzed
and
cytotoxicity
the
sensitivity
absence
in WTd8
cells
5-FU
of WTd
of wild-type
duced by IPTG. As shown
in Fig.
by IPTG enhances
the cytotoxicity
38-fold
of
respond
of wild-
p53
cells
-
+
+
IPTG
-
+
-
+
to 5-FU
expression
in WTd19.
-
in colon
in-
3, induction
of wild-type
of a 20 p.M dose of 5-FU
and by 24-fold
5-FU
p53
by
IPTG-induced
expression
of wild-type
p53 resulted
in a 4-fold reduction
in the
5-FU LD95 dose in both WTd8 and WTd19
(Fig. 3 and Table 1).
IPTG
induction
alter
sensitivity
the cytotoxicity
control
p53
IPTG
of
in GH2
(Fig. 3).
Wild-Type
Topotecan.
hibits
DNA
p53
tothecin
family
in the
below
1
topotecan-induced
p53 induction
has
cytotoxicity
was
modest
compared
topotecan
At concentrations
difference
between
or absence
of
of topotecan,
by the addition
was moderate,
cells and 4-fold
in-
clinical
31). We therefore
cells treated
with
of IPTG.
to
that
of the camp-
shown
is minimal
enhanced
Cells
by stabilizing
cytotoxicity
in the presence
by IPTG.
At higher
concentrations
of IPTG.
exhibiting
in WTd19
How-
Fig. 5 Induction
of DNA fragmentation
in WTd cells.
shows electrophoresis
on 2% agarose
gels of DNA prepared
cells treated
for 24 h with S p.M IPTG or 5 p.M 5-FU,
or
simultaneously.
Molecular
weight
markers:
1353 bp, 1080
603 bp, 310 bp, and 281 bp.
Time
p53
Several
the potentiation
cells.
Potentiates
alternative
by wild-type
When
combining
5-FU
irreversible
growth
arrest
introduction
apoptosis
in some
deleted
or mutated
p53 in the 5W480
assayed
reversible
by DNA
growth
To determine
survival,
cells
induction
the
treated
of wild-type
of
p53
to 5-HI
p53
and
Similarly,
induces
that is consistent
corresponds
to
clonogenic
demonstrates
med after
findings
insufficient
Fig.
a barely
of
of wild-type
cells.
As
results
discussed
in induction
their
were
that,
previous
an
of DNA
argue
to induce
enhances
the cytotoxicity
induction
of apoptosis
that
although
apoptosis
wild-type
in WTd
of 5-Hi
by this
agent.
by
cells,
strongly
p53
the
effects
administration
of
these
administered
wild-type
p53 by
and
of
if instead
of 5-Hi
agents.
Fig.
only
a che-
we first
inves-
6A
timing
shows
of
that
5
p.M
by 48 h of induction
cytotoxic,
whether
agents
the start
potentiation
induced
Wild-
of
relative
these
preceding
minimal
that
by
of
the
of the IPTG treatment
(“concurthe IPTG
treatment
(pre-IPTG).
of administering
is administered
(post-IPTG),
p53,
the
was initiated
at the start
treatment)
or 24 h into
However,
wild-type
varying
two
WTd
combination
for 24 h accompanied
IPTG
were equally
5-Hi
rent”
of
optimal
by 5-Hi
together,
of IPTG
of cytotoxicity
alone.
Thus,
24 h
treatment
was
ob-
potentiation
of
in the cell simultaneously.
Fig. 6B examines
the effects
of combining
24 h of exposure
to 5-Hi
of
p53
after
apopfragto 5
p.M
Fig. 5
examThese
is, by itself,
potentiating
cytotoxicity
along
p53
the
by wild-type
with
IPTG is continued
time of wild-type
modest
IPTG
(Fig.
for
p53
varying
induction
cytotoxicity.
lengths
from
is administered
when
both
the exposure
of time. Shortening
48 to 24 h results
in the degree
magnitude
starting
It is likely
presence
of time after
no significant
that
to
the
in a
of potentiation
of the difference
at the
this
washout
of the drug
difference
is seen
associated
with
IPTG
both
commenced
were
end
effect
of intracellular
cytotoxicity
when
when
be present
between
of a 48-h pulse and a 24-h pulse of IPTG is similar
to
potentiation
of 5-FU
cytotoxicity
observed
when
6A).
continued
but
decrease
The
that both
wild-type
simultaneously,
but demonstrable
the effect
the slight
p53 requires
induction
are begun
of 5-Hi
5 p.M 5-Hi
assayed
by
wild-type
5-Hi
versus
exposures
as observed
fragmentation
finding
that
dose
as
Sensitization
the
clonogenic
with the
that 24 h of exposure
degree
tigated
of
To investigate
agent,
5-Hi,
served
alone did not induce
of observable
DNA
5 shows
Dependence
Type p53
motherapeutic
5-Hi
of
immediately
5 shows
Fig.
observable
decreased
concurrently
examined
figure
induces
a
cycle (28).
survival
of WTd
cells (Fig. 3). In contrast,
marked
DNA fragmentation
in WTd
cells
24-h treatment
of 5-Hi
combined
with IPTG.
strongly
p53,
the p.53 gene has been
induction
of wild-type
not induce
apoptosis
as
5-FU
p.M
IPTG.
with our
approximately
in
for
in WTd
fragmentation
(Fig. 1), but rather
arrest at the Gi phase of the cell
previously,
24 h of IPTG treatment
tosis as determined
by the absence
5-FU
cytotoxicity
induction
tumors
in which
(1 1, 12). However,
WTd
cells does
of wild-type
mentation.
Cell Death
could account
could reflect increased
cell death
cell death
due to necrosis,
or an
the mechanism
treated
with 5
exposure
of 5-Hi
with
decreased
clonogenic
survival
due to apoptosis,
increased
previously,
Apoptotic
mechanisms
p53
The
from WTd8
both given
bp, 872 bp,
an increase
of
cells (Fig. 4 and
1).
Wild-Type
WTd Cells.
24-h
damage
is a member
or absence
ever, this enhancement
only 7-fold in WTd8
Table
WTd
vector
I inhibitor
DNA
colon cancer
(30,
survival
of WTd
there
did not alter
an empty
Sensitizes
which
topOtecan,
also
bearing
Topotecan
drugs,
presence
jiM
did not significantly
itself
cells
and initiates
of
cells
treatment
is a topoisomerase
complex.
activity
against
the clonogenic
in MT2
Modestly
replication
enzyme
either
5-FU
Topotecan
a DNA
drug
of mutant
to 5-FU.
of a 24-h
pulse
is accounted
5-FU
in the cell
of 5-Hi
for
by the
for a period
from the medium.
In contrast,
between
the enhancement
of
a 48-h
versus
a 72-h
simultaneously
exposure
with
Downloaded from clincancerres.aacrjournals.org on July 12, 2017. © 1996 American Association for Cancer
Research.
to
a 24-h
Clinical
Cancer
A
Research
1655
B
1000
1000
No IPTG
-0--
Pro-I PTG
Concurrent
Post-IPTG
100
100
C
C
0
0
U
C.)
Cs
aU.
CS
aU-
0
0
C
C
>
>
10
>
a-
10
Cl)
Cl)
.1
0
0
5
5-Fluorouracil
5
(jiM)
10
20
15
5-Fiuorouracli
25
(jiM)
Fig. 6 Schedule
dependence
of potentiation
of 5-FU cytotoxicity
by wild-type
p53. A, clonogenic
survival
of WTd8
cells treated
with 48 h of IPTG
and with 24 h of 5 p.M 5-FU initiated
concurrently
with IPTG (Concurrent),
24 h into the exposure
of IPTG (Pre-IPTG),
at the completion
of IPTG
(Post-IPTG),
or in the absence
of IPTG
(No IPTG).
B, Clonogenic
survival
of WTd8
cells treated
with
24 h of 5-FU
at the doses
shown.
Simultaneously
with the initiation
of 5-FU, treatment
also was initiated
with IPTG for 24, 48, or 72 h. Control
cells were treated
with 5-FU only (No
IPTG).
A and B show means
of colony
numbers
obtained
in triplicate
experiments;
bars. SE.
exposure
to 5-Hi.
to wild-type
after exposure
that
induction
reversible
This
suggests
that
even
prolonged
exposure
p53 is without
added toxicity
when delivered
to 5-FU, and is consistent
with our previous
of
wild-type
growth
arrest
p53
by
itself
results
well
data
only
in
a
(28).
Discussion
have
plays
a direct
cancer
to
role
more
important
of wild-type
powerfully
chemotherapy,
have
any
continue
in
some
of
p53
been
cell
line
the
control
type
p53
wild-type
effect
promoting
or
in transformed
by
is not,
has
(9).
been
wild-type
cell
types
both
and
not,
similar
cells
by
radiation
p53.
The
role
poten-
(17,
well
approach
32).
cells,
with
resistance
(36).
Our
in
previin
Our
ap-
of
the
wild-type
findings
these
p53
one
cells
are
findings
wild-type
ovarian
p53
cancer
with
cell
line
wild-type
cell
previous
and
p53
in
a direct
Our
consistent
cancer
of
plays
of
an
to
that,
agents.
in
lung
introduction
and
restoring
colon
function
introduction
doxorubicin
however,
in
line,
cells
including
either
that
that
by
in
one
infection
a wild-type
p53 adenovirus
sensitized
the cells to apopkilling
by cisplatin
(33, 35). Our findings
now show that
these
med
to
that
cell
with
totic
report
sarcomas,
finding
therapeutic
wild-
is consistent
demonstrates
wild-type
cancer
findings,
observations
Our
under
that
cells
gliomas,
35).
in a colon
allele
finding
in these
chemotherapy
of
one
Our
sensitizes
and
to
p.53
some
34,
nonetheless
induced
glioma
33,
absence
function
cytotoxic
in
(8,
p53
wild-type
promoter.
itself,
resistance
induction
as
the effects
alleles
18,
to
of wild-type
to examine
p.53
itthe
cells
as
p53
in
the
observations
contrast
line
by
although
repair,
One
can
radiation
of cancer
by the dual
DNA
cells
by
cancer
role
wild-type
expressing
of an inducible
lung
these
Of
reintroduc-
does
cells
mutant
apoptosis
endogenous
cancer
sensitivity
both
taken
that
colon
p53
these
endogenous
promoting
have
on
colon
agents.
to restore
by
with
killing
function
of human
shown
to killing
although
is confounded
inactivating
into
sensitivity
cytotoxic
p53
therapeutic
we have
agents
investigators
cancer
expression
in modulating
putatively
of wild-type
the resistance
of
to express
systems
normal
variety
of p53
cytotoxic
p53
absence
in mediating
enhance
self,
role
that
implication,
and
ous
found
a wide
tion
tial
has
cancer
wild-type
We
cells
proach
wild-type
to
survival
also
in colon
new
5-FU,
radiation,
p53
p53
several
cancer
classes
and
topotecan.
of colon
cells
in the
a wide
dose
range
over
allow
us
to
of
quantitate
cell
line
sensitizes
therapeutic
agents,
As we have
presence
or
of cytotoxic
the
magnitude
exam-
absence
of
agents,
our
of
Downloaded from clincancerres.aacrjournals.org on July 12, 2017. © 1996 American Association for Cancer
Research.
the
1656 Wild-Type
p53 Potentiates
Cytotoxicity
sensitization
induced
by
dose enhancement
factor
ment
p53 both in terms
of a
as a cytotoxicity
enhance-
shown
in the
WTd
results
in expression
rable
previously
derivative
to that
of the
5W480
p53
demonstrate
recent
colon
gene
levels
p53
demonstration
type
the
novel
alleles
p53
cell
wild-type
some
sarcoma
and
line
generality
some
function
effects
either
of inactivating
in normal
med.
Different
methods
use of cells
from
of human
cells
of
mutant
with
virus
to many
alleles.
These
cell
and
the
found
chemotherapeutic
sensitivity
further
contrast,
human
papilloma
to
p53
virus
included
wild-type
was
the
cells
into
yielded
con-
from
p53
In contrast,
no
(24).
p53
by introduction
in human
to cisplatin
(17),
breast
cancer
cell
cells
to cisplatin
ings
is that the functional
alleles
may differ
p53
transformed
formed
p53
alleles
been
sensitize
resolution
or may
differ
when
tolerate
the
continued
and transformed
eliminated.
is consistent
colon
with
that
cells
Our
cancer
studies
the
cells
in naturally
presence
alleles
p53
therapeutic
occurring
of mutant
agents
human
alleles
p53
with resistance
with resistance
to cisplatin
to cisplatin
(22, 23); in lung
(38); in gastric
cancers
is associated
with
to multiple
chemotherapeu-
resistance
One
reason
the
approach
of reconstituting
wild-type
more
stable
wild-type
p53
function
Certainly,
wild-type
p53
directly
including
hematopoietic
and some
However,
is restored.
induces
apoptosis
cells
(1 1, 12, 40),
squamous
carcinomas
this property
is clearly
p53
widely
used is the
cell lines in which
restoration
in many
some
of the head
not universal,
S., Fearon,
4. Kastan,
Science
E., Nigro,
and Ms.
radiation
B., and
(Washington
J., Hamilton,
P., Ledbetter,
D., Barker,
M., Onyekwere,
R. Participation
experi-
DC),
Harris, C. p53
253: 49-53,
suppressor
S., Preisinger,
Sidransky,
of p53 protein in the cellular
51: 6304-631
1, 1991.
Res.,
tumor
A., Jessup,
K., Nakamura,
17 deletions
(Washington
0.,
p53
Y., White,
R.,
and p.53 gene mutations
DC), 244: 217, 1989.
in
D., Vogelstein,
B., and Craig,
response
to DNA damage.
M. B., Zhan,
Q., El-Deiry,
W. S., Carrier,
F., Jacks,
T.,
Walsh,
W. V., Plunkett,
B. S., Vogelstein,
B., and Fornace,
A. J., Jr. A
mammalian
cell cycle checkpoint
pathway
utilizing
p53 and GADD45
is defective
in ataxia-telangiectasia.
Cell, 71: 587-597,
1992.
Sci.
USA,
L.,
B., Walsh,
W., and Kastan,
determinant
following
89: 7491-7495,
1992.
Kassel,
J.,
M., Bressac,
8. Mercer,
W.,
Nelson,
C.
B., Ozturk,
Shields,
J., and Ullrich,
E.,
M., Amin,
S. Negative
9. Hartwell,
L., and Kastan,
(Washington
DC),
266:
M.
Litwak,
0.,
11. Ramqvist,
T., Magnuson,
Wild-type
induces
apoptosis
p53. Oncogene,
of
cell
types,
gliomas
(41),
and neck (42).
and, as men-
p53
mutant
Yonish-Rouach,
cells
that
in osteosarco-
Appella,
wild-type
control
E., Romano,
p53.
tumor
Proc.
and cancer.
Y., Szekeley,
in a Burkitt
8:
E., Resnitzky,
is inhibited
G.,
B., and
Natl.
Science
1994.
K., Wang,
A., and Oren, M. Wild-type
p53
Natl.
in a glioblastoma
human
1990.
M. Cell cycle
A.,
protein
regulation
1821-1828,
Proc.
S. J., Vogelstein,
M., Sauve,
growth
M. Wild-type
irradiation.
Gryka,
M., Baker,
cell line that conditionally
expresses
Acad. Sci. USA, 87: 6166-6170,
D.,
p53 induces
by interleukin-6.
L., and Klein,
lymphoma
1993.
1495-1500,
Lotem,
apoptosis
Nature
T. p53 is
Nature
(BL)
J., Saches,
L.,
of myeloid
(Lond.),
line
352:
G.
that
Kimchi,
leukaemic
345-347,
1991.
13.
with
agents
(39).
function
in tumor
cells has not been
experimental
difficulty
in constructing
Donover,
and
D., Vogelstein,
cancers.
and Vogelstein,
B. Chromosome
colorectal
carcinomas.
Science
12.
is associated
is associated
(20); and in Burkitt’s
lymphomas
is associated
to radiation
(2 1 , 39) and to chemotherapeutic
3. Baker,
J., VanTuinen,
carries
tumors,
in ovarian
cancers
cancers
tic agents
resistance
gene
p53
10. Lowe,
S., Schmitt,
E., Smith,
S., Osborne,
B., and Jacks,
required
for radiation-induced
apoptosis
in mouse
thymocytes.
(Lond.), 362: 847-849,
1993.
alleles
p53
wild-type
to cancer
trans-
of wild-type
wild-type
that
topotecan
Friend,
S. H. p53 functions
as cell cycle control
mas. Mol. Cell. Biol.,
10: 5772-5781,
1990.
wild-type
normal
and
between
Mr. P. Scott
in the
M., Sidransky,
in human
7. Diller,
Gabhardt,
find-
presence
in which
finding
1. Hollstein,
Acad.
conflicting
compared
combination
ments.
of
fibro-
in the
of the
consequence
of inactivating
when
compared
between
in
the
therapy.
Chatterjee,
assistance
6. Kuerbitz, S., Plunkett,
is a cell cycle checkpoint
(24); and
sensitivity
of these
that
SW480
suggesting
One
cells,
cells
have
(17).
but not to radiation
line to increase
the
for
In
blasts
to sensitize
cells to multiple
chemotherapeutic
agents
(1 8); in the RKO colon cancer cell line to increase
the sensitivity
of cells
MCF-7
studies
determining
5. Kastan,
non-
showed
shown
Dr. Satadal
E. Swinler
Cancer
to radiation
chemotherapy
potential
or radiation
Future
in
A., Momand,
J., and Finlay,
C. The
gene. Nature (Lond.), 351: 453-456,
1991.
exam-
into
mice
of
(34).
helpful
I 991.
to radiation-induced
animals
when trans(14).
or
E6 genes
have
have
knockout
radiation
inactivating
been
to be resistant
agents
from
have
Lymphocytes
efficacy
cells
be
2. Levine,
alleles,
introduction
methods
types.
and Ras were
fibroblasts
in
studies
cancer
will
References
in which
p53
the introduction
were shown
to be resistant
and fibroblasts
from these
E1A
transformed
change
these
mice,
E6 genes,
in different
mice
(10),
formed
by
types,
lung
types
chemo-
We thank
Sandra
mutations
wild-type
cell
knockout
p.5.3
results
knockout
apoptosis
and
used
p53
papilloma
flicting
endogenous
or in transformed
can
of wild-
p53 function
in human
tumor cells.
This study contrasts
with previous
investigations
the
with
and
cell
in
in
Acknowledgments
not depend
on
p53 in cancer
measure
(33)
other
wild-type
p53 induces
growth
arrest
and induces
only growth
inhibition
Thus,
that bind to C-terminal
that it will be possible
for restoring
restoring
cells (8)
of
therapy
of combi-
p53
above,
glioma
these
compa-
(28).
efficacy
therapy
does
of wild-type
that
methods
cancer
at levels
the potential
be restored
in 5W480
cells by antibodies
epitopes
in p53 (37) gives encouragement
to develop
of wild-type
protein
mutant
that
nation chemotherapy
with
achieving
supraphysiological
The
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Wild-type p53 protein potentiates cytotoxicity of therapeutic
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Clin Cancer Res 1996;2:1649-1657.
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