Vol.

4, 467-473,

1993

June

Cell Growth

Reconstitution
of Wild-Type
Apoptosis
in a p53-negative
induced
T-Cell Lymphoma

Visong

Wang,2

Torbj#{246}rn Ramqvist,

H#{226}kan
Axelson,

George

Department
of Tumor
01 Stockholm,
Sweden

Klein,

Biology,

Laszlo

tutes

Szekely,

Institute,

Inactivation

or mutation

S-104

variety

transformed

of human

and murine tumors. We have found that a v-myc

retrovirus (J3)-induced T-cell lymphoma line (J3D) has
lost one of its p53
become
inactivated

murine

alleles,
whereas
the other has
due to the insertion
of a Moloney

leukemia

provirus

in intron

4 with an opposite

transcriptional
orientation. No p53 protein could be
detected by immunoprecipitation
with monoclonal
anti-p53

antibodies.

We have transfected

the temperature-sensitive

murine Va1135 construct that

number of viable cells among the p53 transfedants,

parental

cells, and neomycin

at 37#{176}C.
Following

vector-transfected

a temperature

shift

the

control

from

p53-transfected

cells

grown

at 32#{176}C

revealed nucleosomal
fragmentation,
indicating
cell
death by apoptosis.
It is suggested that apoptosis is

triggered by contradictory
signaling. Constitutively
expressed v-myc can stimulate cell proliferation,
whereas

expression

lost endogenous

neoplastic

of wild-type

p53

p53 in cells that have

expression

development

in the course

of their

may suppress growth.

p53 gene is one of the best characterized

tumor
suppressor
genes (for reviews,
see Refs. 1 and 2). Inactivation
of the p53 gene by point
mutations
or gene
deletion,
truncation,
or nonsense
mutation
has been
detected
in a wide variety
of human malignancies
and is
The

the

most

man cancer

(3). In the mouse,

Received
1/25/93;
revised
i This
work
was supported
National
Cancer
Institute,

common

Research,

grant from
2 To whom
Tumor
Sweden.

alteration

retroviral

insertion

3/17/93;
accepted
3/17/93.
by a USPHS
Grant
CA14054
Department
of Health
and

and grants
from the Swedish
Cancer
Bergvalls
Stiftelse.
Y. W. is supported
Research
Institute,
New
York,
NY,

Cancer

genetic

Los Angeles,

in hu-

consti-

awarded
by the
Human
Services,

Society
(Cancerfonden)
and Magn.
by fellowships
from
the Cancer
and the Concern
Foundation
for

CA. K. G. W. is the recipient

Karolinska

Institute,

Box

60400,

S-104

inactivation,

as dem-

virus-induced
erythroleumurine
leukemia
virus(7).

p53

can

also

be

macti-

p53

(13,

16).

Expression

of exoge-

wild-type
(24)

p53

induced

or human

colon

wild-type

In this study,
negative

apoptosis

carcinoma

cells

myeloid

(25)

that

cells

lacked

p53.

we have analyzed

I-cell

in mouse

lymphoma

line,

the p53
J3D,

gene

carrying

in a p53a constitu-

v-myc gene. We show that one p53 allele

by retroviral
insertion
and that the other
allele is lost. In order to examine
the significance
of loss
of wild-type
p53 in the J3D cells, we have transfected
the cells with a temperature-sensitive
VaIl 35 p53 mutant
that is expressed
largely
as mutant
p53 at 37#{176}C,but
predominantly
as wild-type
p53 at 32#{176}C
(26). We have
found
that expression
of exogenous
wild-type
p53 at
32#{176}C
in this line elicits substantial
cell death through
apoptosis.
We suggest
that apoptosis
is triggered
by
tively

activated

is inactivated

signals:

stimulation

of cell

proliferation

by

a constitutively
active v-myc, and growth suppression
by
wild-type
p53 in a cell line whose tumorigenic
development has included
loss of wild-type
p53.
Results

Characterization
of the J3D Line. The original
J3Dori
lymphoma
was Thy-i
positive.
The cell line (j3D) derived
from it did not express
Thy-i,
CD4, CD8, CD3, B220,
immunoglobulin,
Mac-i,
or Mac-2.
J3D cells were also
negative
in the peroxidase
reaction
test. The J3Dori tumor and the J3D cell line showed
the same TCR3-y
rearrangements
on Southern
blots (Fig. 1 ), whereas
the
TCR-f
chain and the immunoglobulmn
heavy
chain
region were in germlmne configuration
(data not shown).

of a personal

the Concern
Foundation
for Cancer
Research.
requests
for reprints
should
be addressed,
at Department

Biology,

p53

leukemia

wild-type

contradictory

Introduction

considered

for

nous wild-type
p53 was also shown
to promote
differentiation
of p53-negative
mouse pre-B leukemia
(22) and
human chronic
myeloid
leukemia
cells (23). In addition,

endogenous

to 32#{176}
C,

the p53 transfectants rapidly lost viability, and 95100% of the cells were dead by 3 days, whereas the
control cells remained unaffected. Examination of DNA
isolated

pre-B

exogenous

this line with

is expressed
as mutant
p53 at 37#{176}C
and largely
wildtype p53 at 32#{176}C.
There was no difference
in the

cells

mechanism

vated by complex
formation
with transforming
proteins
of DNA tumor viruses, including
the SV4O large I antigen
(8, 9), the adenovirus
El B protein (10), and the E6 protein
of the oncogenic
subtypes
of human
papilloma
viruses
(1 1). E6-p53 complexing
is followed
by rapid degradation
ofp53
(12).
Introduction
of wild-type
p53 inhibits
the growth
of
tumor cells that lack endogenous
p53 or carry mutant
p53 (13-20).
Wild-type
p53 has been shown to induce
growth
arrest at the G1 phase of the cell cycle (14, 21).
Cells expressing
endogenous
wild-type
p53 appear
less
sensitive
or resistant
to the growth-inhibitory
effect of

of the p53 tumor suppressor

in a wide

another

onstrated
in Friend leukemia
kemia
(4-6) and an Abelson

Abstract

gene has been observed

467

p53 Expression
Triggers
v-myc RetrovirusLine

and KIas G. Wiman

Karolinska

& Differentiation

01

Stockholm,

of

The

abbreviations

temperature-sensitive;
loney

murine

leukemia

used

are:

cDNA,
virus

TCR,

T-ceIl

complementary
long

terminal

receptor;

DNA;
repeat;

kb,

kilobase(s);

Mo-MuLV-LTR,
bp,

base

pair(s).

ts,

Mo-

468

p53-triggered

Apoptosis

in v.myc-induced

Cell

Line

B
2

v-myc-28S
I.

I.

-18S

6.2 kb

-28S
actin4.0kb

-18S

Fig. 2.
Northern
blot analysis
of v-myc
expression.
Total
RNA
(10 g)
was analyzed
by blot hybridization
with a 1 .5-kb v-myc
probe.
The same
filter was stripped
and rehybridized
with a 0.9-kb
a-actin
cDNA
probe,
as a control
for the amount
of applied
RNA.
A, J3D cells;
B, normal
kidney.

4.1kb-

-.

of the rearrangement,
tially hybridized
with
(Fig.
with
BamHl

EcoRl

1. Southern
blot
analysis
of the TCR-y
chain
gene.
A, BamHldigested
DNA was hybridized
with a TCRprobe.
Note
the rearranged
4.1-kb
fragment.
B, EcoRl-digested
DNA was hybridized
with the TCR-
probe.
Note
that
the
16.kh,
6.2.kh,
and 4.0-kb
bands
represent
rearranged
fragments.
Lane
1, l3Dori
lymphoma
cells:
Lane 2, j3D cells;
Lane 3, normal
kidney.
fig.

48), whereas
the RH7 and

fragments

could

Kpnl-digested

PP5 probe,
and

two

copies
not

v-myc
mRNA
the cells were

of cytogenetically

shown),

on

which

normal
the

(Fig. 2). Karyotype

diploid
with
two

chromosome

mouse

p53

gene

1 1 (data
has

been

localized
(27). Ten immunodeficient
mice were used for
tumorigenicity
tests. All mice developed
donor-derived
tumors
12-15
days after inoculation,
as judged
by the
identity
of the v-myc
bands between
the tumors
and the
inoculated
J3D cells on Southern
blots (data not shown).
Alteration
of p53 in J3D Cells. In order to assess the
expression
of p53 in J3D cells,
[35S]methionine-labeled

cell extracts

were

immunoprecipitated

antibodies
(PAb421),

directed
against
both
only mutant
(PAb24O),

(PAb246).

As shown

with
wild-type
or only

in Fig. 3, no p53 was immunoprecip-

itated
from
j3D cells with
any of
PAb421
antibody
precipitated
the
from
the
Rauscher
virus-induced

(RMA)

monoclonal
and mutant
wild-type
p53

the antibodies.
The
53 kilodalton
band
lymphoma
cell line

used as control.

to examine
the status of the p53 gene in J3D
cells, Southern
blot analysis was performed
with EcoRldigested
DNA and probed
with RH7, a genomic
exon 1
fragment
(Fig. 4B). This probe
detected
a germline
band
of 15 kb in the original
J3Dori
lymphoma
cells and in
normal kidney cells. In J3D cells, the same probe hybridized with a new 21-kb band and failed to identify
any
15-kb germline
fragment
(Fig. 4C). For further definition
In order

There

4D).

(data not shown).

DNA

rearranged

1 .1 kb in size.

only

the

To investigate
cells expressed
showed
that

be detected

was

However,

hybridized

fragments

was

was hybridized
No rearranged
with

appeared,

no germlmne

4.7-kb

the

4.0 kb
fragment

(Fig.
It indicates
that novel Kpnl sites were generated
in the region defined
by the PP5 probe. The XP1O probe
detected
only the 4.0-kb band, and the PK3 probe detected

The J3D
analysis

BamHI-digested
DNA
KH6 probes
separately.

when

the

Kpnl-digested
DNA was sequenthe RH7, KB4, and KH6 probes

tion

fragments

insertion,

1.1-kb

band

(Fig.

the possibility
might

have

been

the same Kpnl-digested

and rehybridized
with
probe.
It only hybridized

a Mo-MuLV
with the

that was also recognized

not with
the
xP1O probes

4.0-kb
(Fig.

4D).

that the novel

pS3 restric-

generated

by

DNA

was stripped

filter

U3
novel

retroviral

LTR (MoU3LTR)
1.1-kb
fragment

by the PP5 and PK3 probes,

fragment

4D). The

detected
novel
21-kb

but

by the PP5 and

p53 EcoRl frag-

ment also hybridized

with the MoU3LTR
probe, but not
with the v-myc
probe (data not shown).
These results
suggest that a Moloney
proviral
fragment
has been inserted
into intron
4 of the p53 gene within
the region
defined
by the PP5 probe, as indicated
in Fig. 4A. Since
KpnI sites are present
only in the US portions
of the MoMuLV LTR sequences
(28), hybridization
of the 1.1-kb
fragment
to both the MoU3LTR
and PK3 probes indicates
that the provirus
has been inserted
in an opposite
transcriptional
orientation
compared
to the p53 gene.
Reconstitution
of J3D Cells with Wild-Type
p53. The
J3D cells were cotransfected
with the temperature-sensitive

and

mouse

the

pCMVneoBam

Val135

neomycin
(13).

mutant

p53

resistance
G418-resistant

construct

(ts p53)

gene-carrying
cells

were

(26)

plasmid
single

cell

cloned.
Clonality
was confirmed
by the detection
of
unique
integration
sites on Southern
blot analysis with a
PS3 probe (data not shown).
Six clones that carried
ts
p53 integrated
at different
sites were subjected
to further
analysis.

Cell

37#{176}C

234

32#{176}C

56

200-

97-

Discussion

explained
by chromosome
loss, as cytogenetic
examination
showed
that the cells contained
two normal
chromosomes
1 1 (data not shown).
Since the original
J3Dori

69-

p53-

tumor did not show any p53 gene rearrangement,

the
insertional
inactivation
of one p53 allele, and the loss of
the second allele, must have taken place during the in
vitro establishment
of the j3D clone. Alternatively,
the
J3D cell might have been present
in the original
in vivo

46-

tumor

,,

Cl

C4

?
C#{176}

Fig. 3.
Immunoprecipitation
of p53
transfectants.
Equal
amounts
of cell
immunoprecipitated
with
p53-specific

C4

Cl

C4

in parental
J3D cells
and ts p53
lysates
from
each
sample
were
monoclonal
antibodies
as mdi-

cated
below
each lane. Lane 1, RMA;
Lanes 2-4, J3D parental
cells; Lanes
5 and 6, J3DVaI135M3
cells grown
at 37#{176}C;Lanes 7 and 8, J3DVaI135M3
cells after 24 h at 32#{176}C.

The expression
of p53 in the six clones
at 37#{176}C
and 32#{176}C
by immunoprecipitation
monoclonal
antibodies
PAb24O
(mutant
PAb246

(wild-type

specific).

As

was evaluated
using the
specific)
and

shown

in

Fig.

3, both

wild-type
and mutant
p53 were detected
at both temperatures
in a representative
clone, J3DVaI135M3,
but
the ratio of PAb246to PAb24O-reactive
p53 is significantly
higher after 24 h at 32#{176}C.
This is in agreement
with previous
studies
(26). Essentially
identical
results
obtained

with

six individual

Expression
of Wild-Type
Apoptosis.
The
viability

clones.
Cells Triggers
transfectants,

At 32#{176}C,however,

the

ts p53

transfectants

rapidly

lost viability.
After 3 days, 95-100%
of the cells were
dead (Fig. 5). The pCMVneoBam
transfectants
and parental cells remained
unaffected
at 32#{176}C.
The

ts p53-transfected

densation

in one

shown)

cells

or several

at 32#{176}C,indicative

transfectants,

was intact.

chromatin

con-

fragments

(data

of

death

apoptosis

(29). This was confirmed

by the
teristic
oligo-nucleosomal
DNA
in these cells (Fig. 6). The DNA,
the ts p53 transfectants
grown at
control

showed

nuclear
cell

by

not

presence
of the characfragmentation
ladder
examined
in parallel,
of
37#{176}C,
of pCMVneoBam

or of parental

cells

grown

at 32#{176}C

gained

undetectable

the

upper

hand

by Southern
during
in

blot
vitro

It has been
shown
that constitutive
c-myc
can trigger
apoptosis
in rat fibroblasts,
when cell growth
is inhibited
by serum depletion,
isoleucine
starvation,
or thymidmne
block, at various stages during the cell cycle (32). Similarly, constitutive
c-myc accelerated
apoptosis
in an interleukin
3-dependent
mouse
myeloid
cell line upon
interleukin
3 withdrawal
(33). Taken together,
these resuIts indicate
that apoptosis
may be triggered
by contradictory
signals.
Activated
myc is known
to stimulate
cell
proliferation.
If this occurs concurrently
with the transmission of strong growth-arresting
signals, the cell may
respond
by initiating
an apoptotic
program.
Expression
of wild-type
p53 has been shown to arrest the cell at the
G1 phase ofthe cell cycle in most cell systems tested (14,
21) and could provide
such a signal. Recently,
it was

reported

that

myeloid
derived
induced
found to
(34).

It

reexpression

of wild-type

p53

in a mouse

leukemia
cell line and a human
colon tumorcell line that lack endogenous
p53 expression
apoptosis
(24, 25). p53-mediated
cell death was
occur preferentially
in G1 or at the G1-S bound-

amy, although

wild-type

p53

will be interesting

did

not

induce

to determine

notion

that

eliciting
apoptosis
ments demonstrating
by antisense

c-myc

can

play

receives
further
that inhibition

oligonucleotides

blocks

growth

whether

of myc (enforced
entry
into S phase)
(G1 arrest) is dominant
in our system.
The

p53 in the J3D

of the
ts p53

J3Dneo controls,
and the parental J3D cells was evaluated
at 37#{176}C
and 32#{176}C.
At 37#{176}C,
all cell lines grew equally
well.

as a subpopulation

that
later
cultivation.

30-

were

469

& Differentiation

No p53 protein
was detected
in J3D, a cell line established from a v-myc
retrovirus-induced
murine
I-cell
lymphoma,
J3Dori.
Our molecular
analysis
has shown
that one p53 allele has been lost, whereas
the second
has been inactivated
by insertion
of a Moloney
provirus
into intron 4 with an opposite
transcriptional
orientation,
presumably
preventing
appropriate
processing
ofthe fulllength
p53 transcripts.
It is noteworthy
that an intact
intron 4 has been shown to be essential for p53 expression (30, 31). The loss of one p53 allele could not be

Growth

arrest

the effect

or wild-type

an

p53

important

role

in

support
from experiof c-myc expression
activation-induced

apoptosis
in I-cell
hybridomas
(35). If apoptosis
is induced
in the J3D line by the contradictory
myc versus
wild-type
p53 signals, it should
be preventable
by specific

inhibition

of myc

expression.

Would

the

induced

expression
of wild-type
p53 at 32#{176}C
cause reversible
growth arrest, rather than apoptotic
death, if myc expression was inhibited
by antisense
RNA or oligonucleotides?
Overexpression
of bci-2 may also counteract
the apoptosis-mnducing
signals, in analogy with recent findings
(36,
37).
Materials
and Methods
The T-CeII Lymphoma
Line J3D. T-cell lymphomas
induced
in newborn
(AKR x BALB/c 6;1S)F1 mice

were
by J3,

470

pS3.triggered

in

AP0PtO515

Cell

induced

Vffl5(

Lii

El

E3

E6E8

E2 El

ES El

i-i-

A.

E9 El DEll

uI1IIIII

-.

.-

.-

M.-MuLY

Fig.

4.

mouse

B
RH

P
I

XHK
,PB
Iiilliui

PB

PPX

PFK1

1111111

xp1o

RH?

KH

Ill

PK3
pp5

-L

KH6

K84

II

1 kb
C

Eco RI

KPnIp

21 kb
15kb

4.0 kb
.

Probes:

a v-myc-carrying

of

the

character-

0.

xP10

RH7

retrovirus,

and

U3LTR
probes.
Two novel
bands
of 4.0 kb and 1.1 kb were
detected
by the PP5 probe
in Lane
2. XP1O detected
only
the 4.0kb fragment,
and PK3 hybridized
only
with
the
1.1-kb
fragment.
The 1 . 1 .kb fragment,
but not the
4.0-kb
fragment,
hybridized
with
both
the
p53
and
MoU3LTR
probes.
Lane
1, J3Dori
lymphoma
cells;
Lane 2, l3D cells;
Lane 3, normal
kidney.

4.11th

1.1

map

I gene

ization
of p9 1 gene
in l3D cells
using p53 and MoU3LTR
probes.
A, exons as determined
by Bienz
et a!. (53( are depicted
as solid
boxes.
The
approximate
inserlion site of the Moloney
murine
leukemia
Provirus
is indicated;
arrow;
the transcriptional
direclion of the provirus.
B, the cleavage sites for the restriction
enzymes
EcoRl
(RI,
Hindlll
(H),
Pvulf
(P(, Xhol
(X(, BamHl
(B(,
and Kpnl (K) are indicated.
Bars,
probes
used.
C, EcoRl.digested
DNA
was
hybridized
with
the
RH7
probe,
corresponding
to
p53 exon
1. A 21-kb
rearranged
fragment
is visible
in Lane 2. D,
DNA was digested
with Kpnl and
consecutively
hybridized
with
the XP1O,
PP5, PK3,
and
Mo-

Physical
p

propagated

PP5

in the presence

of Moloney
murine
leukemia
virus helper
(38). The viruscontaining
supernatant,
received
from
Dr. Ulf R. Rapp
(National
Cancer
Institute,
Frederick,
MD),
was inoculated
i.p. The original
J3Dori
lymphoma
arose
in the
spleen
of one mouse
66 days after virus infection.
Singlecell suspensions
were
prepared
from
the splenic
lymphoma
and
cultured
in RPMI
1640
medium
supplemented
with
10%
fetal calf serum.
The J3D cell line was
established
from the suspended
cells after 1 month
cultivation
in vitro. For tumorigenicity
tests, S x 106 J3D cells
were
inoculated
s.c. or i.p. into immunodeficient
(ACA
x CBA)F,
mice pretreated
with 200 mg/kg cytosine-$-Darabinofuranoside
for 3 days, followed
by 735 rad wholebody
irradiation.
Chromosome
analysis
was performed
as described
previously
(39).

MOU3LTR

PK3

Staining
and Enzymatic
Analysis.
Expression
of surface
markers
was assessed
by immunostaining,
using antibodies reacting
with Thy-i,
CD4,
CD8,
CD3,
B220, Mac-i,

Mac-2,
and immunoglobulmn.
Peroxidase
reaction
with
benzidine
was performed
as described
(40). To detect
apoptotic
cell death, the cell smears were prepared
by
cytospin
and stained
with
May-Grunwald-Giemsa.
bility
was assessed
at different
time
points
mocytometer
and trypan
blue exclusion.
Plasmids
and Transfedion.
The plasmid

GVaI13S,
meric
DNA,

or temperature-sensitive

gene, consisting
of mouse
p53
including
introns
2-9,
under

control

of a Harvey

It encodes

for

p53,

alanine

a mutant

sarcoma
protein

at position

135

virus
with

(41,

using

Viaa he-

pLTRp53c-

carries

a chi-

cDNA
and genomic
the transcriptional

long

terminal

a substitution

42).

At

repeat.
of valine

37#{176}C,it can

Cell Growth

& Differentiation

471

100
90
80
70
60

so
40
30
20
Fig. 5.
Viability
control
cells and
32C.
Cells were
cells/mI
in RPMI

of J3D parental
cells,
J3Dneo
0
ts p53 transfectants
at 37C
and
8
seeded
at a density
of 4 x 10#{176}
1640
medium
containing
10%
.0
fetal calf serum
and incubated
at 37#{176}C(A) and
32C
(B). Viability
was determined
at each
time
point
by Irypan
blue exclusion.
The results
represent the mean
viability
of quadruplicate
cultures.
Black
boxes,
parental
J3D
cells;
open
boxes,
l3DneoM3
cells; black circles,
J3DVaI135M3
open circles,
I3DVaI135M8
cells.

10

-.

0
0

10

20

30

40

50

60

10

20

.-

70

If

cells;

30

40

50

60

70

Hours

transform
mutated
formation

rat

embryo

fibroblasts

in cooperation

ras. The protein

assumes
at 32#{176}C
and can suppress

transformation
the neomycin

with

largely
wild-type
conrat embryo
fibroblast

(26). The plasmid

pCMVneoBam
carries
resistance
gene driven by the herpes sim-

plex virus
thymidine
kinase
promoter
(13). pLTRp53cGVali 35 and pCMVneoBam
were cotransfected
into J3D
cells by electroporation
using a Bio-Rad
Gene
Pulser set

at 250 V and
transfected

960 zF. As a control,

alone.

G4i8-resistant

pCMVneoBam
cells

were

cloned

was
by

heavy chain J region (46). The MoU3LTR

corresponds
to the U3 region of the Mo-MuLVLTR
(47).
p53 probes
included
RH7, a 700-bp
EcoRlHindlII
exon 1 genomic
fragment
(48); XP1O, a 1.0-kb
XhoI-PvulI
fragment
covering
a region from exon 2 to the
first Pvull site in intron
4; PP5, a 500-bp
PvuIl-Pvull
fragment
corresponding
to the 3 part of intron 4 and 33
bp into exon 5; PK3, a 300-bp Pvull-Kpnl
fragment
coyering sequences
in exon 5, intron 5, and part of exon 6,
5 of the Kpnl site; KB4, a 400-bp
Kpnl-BamHI
fragment
immunoglobulmn

probe

limiting dilution.
Clonality
was assessed by Southern
blotting, using Kpnl-digested
DNA and a p53 genomic
probe
(KH6) that contains
exon 9 (Fig.
DNA
and RNA Analyses. High-molecular-weight
DNA

covering
the
bp Kpnl-Hindlll

was

blotting
from the cloned
chicken
MC29 virus (49) was used as a
v-myc
probe. The murine a-actin
probe is a 0.9-kb PstlPstI cDNA
fragment
(50).
Immunoprecipitation.
Mutant
and wild-type
p53 pro-

4B).

isolated

from

cells

by

conventional

methods

(43).

zg of DNA were analyzed

by Southern
blot hybridization after BamHl,
EcoRl,
Hindlll,
and Kpnl digestions
and electrophoresis
on 0.8% agarose gels. The following
TCR and immunoglobulmn
gene probes were used: a 1.6kb fragment
of the mouse
TCR-y constant
1 .2 chain
containing
the VJC region (44); a 5.6-kb BamHI-Hindlll
and a 2.0-kb EcoRl-EcoRl
fragment
of the mouse TCR-3
chain
CiA
containing
the jfll + Cfli region and the J32
region,
respectively
(45); and the p12/23JH
probe contaming
a 2.0-kb
BamHl-EcoRI
fragment
from the mouse
Ten

sequences
v-myc

teins
ously
PAb421,

3 end

6, and

intron

6; KH6,

a 600-

to exon

9 and

9 (Fig. 48).
mRNA
expression
was determined
by Northern
as described
(43). A 1 .5-kb PstI-PstI
fragment

were
detected
by immunoprecipitation
described
(51), using
the monoclonal

and

as previantibodies

and PAb246 obtained

from Oncogene
York, NY). PAb421 recognizes
both mutant
p53 proteins,
whereas
PAb24O reacts with

PAb24O,

Science (New
and wild-type
mutant

of exon

fragment
corresponding
5 of the HindIIl
site in intron

PAb246

wild-type

p53

only

(52).

472

p53.friggered

Apoptosis

in v.rnvc-indu(

Marker

((I Cell

Line

Ben-David,
tional
inactivation
for identifying
1990.

Y., Lavigueur,
A., Cheong,
of the p53 gene during
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6. Hicks,
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M. Integration
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Virol.,
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7.

Wolf,
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G. Y., and Bernstein,

A. InserFriend
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murine
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D. , and Rotter,
V. Inactivation
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8. Lane, D. P., and

in SV4O-transformed

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1984.
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9. Linzer,
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Howley,
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12. Scheffner,
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M.,
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fragmentation
analysis.
Cells
were
collected
at different
time
p(iints,
and
DNA
was extracted
as described
in Materials
and
Methods.
Each DNA sample
of 3 g was run on a l.5%
agarose
gel and
stained
with
ethidium
bromide.
Molecular
sizes in base pairs of marker
DNA
fragments
(BRL,
Gaithersburg,
MD)
are
indiated.
Lane
1, (3D
Parental
(ells at 37C;
L,ine 2, (3DVa1135M3
(ells
at 37C;
Lane
1, (3D
parental
cells after 48 h at 32C:
Lane 4, l3DneoM3
cells after 48 h at
32C;
L,ines 9-8,
(3DVaI135M3
cells alter 6, 15, 24, and 48 h at 32C,
respectively.

Electrophoretic
Analysis
of DNA Fragmentation.
Cells
grown
at 37#{176}Cor 32#{176}Cwere
harvested
at various
time
intervals
and washed
with
phosphate-buffered
saline.
Cells were
incubated
at 37#{176}Cfor 6 h in lysis buffer
(50
mM
Tris, pH8-i00
mM
EDTA-100
ms NaCl-i%
sodium
dodecyl
sulfate-50
og/ml
proteinase
K). The DNA
was
then extracted
with phenol-chloroform
and precipitated
in ethanol.
Each DNA
sample
of 3 zg was electrophoresed through
a 1 .5% agarose
gel and stained
with ethidium bromide.
Acknowledgments
We

thank

Dr.

Moshe

Oren

for the

gift

of the

Bert Vogelstein
for the gift of the pCMVneoBam
to acknowledge
M. Babonits,
M. L. Solberg,
technkal
assistan(
e.

p53VaIl

35 plasmid

and

plasniid.
We would
and M. Hagelin
for

Dr.
like
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