(CANCER RESEARCH57. 1895-1902. May 15. 19971
DNA Damage-associated
Dysregulation
of the Cell Cycle and Apoptosis Control in
Cells with Germ-line p53 Mutation'
Kumiko Goi,2 Masatoshi Takagi, Satoshi Iwata, Domenico Delia, Minoru Asada, Rosangela Donghi,
Yukiko Tsunematsu, Shinpei Nakazawa, Hiroshi Yamamoto, Jun Yokota, Kazuo Tamura, Yoshifumi Saeki,
Joji Utsunomiya, Takashi Takahashi, Ryuzo Ueda,3 Chikashi Lshioka, Mariko Eguchi, Nanao Kamata,
and Shuki Mizutani4
Department of Virology. The National Children ‘sMedical Research Center, 3-35-31 Taishido, Setagaya-ku, Tokyo 154. Japan (K. G.. M. T., S. I.. S. M., M. Al: Division
Oncologia Sperimentale. !nstituto Nazionale Tumori, Via G. Venezian 1. 20133 Milan. italy ID. D.. R. DI: Department of Hematology. The National Children s Hospital. 3-35-3!
Taishido, Setagaya-ku, Tokyo 154, Japan (1'. TI: Department of Pediatrics, Yamanashi Medical University, 1110 Shimokawahigashi, Tamaho-cho. Nakakuma-Gun, Yamanashi
409-38, Japan (S. NJ; Department of Surgery, National Cancer Center Hospital, 5-1-! Tsukiji. Chuo-ku 104, Tokyo, Japan (H. Y.J: Biology Division. National Cancer Center
Research Institute, 5-1-1 Tsukiji, Chuo-ku 104, Tokyo, Japan (J. Y.J: Department of Surgery. Hyogo Medical University. 1-1 Mukogawa. Nishinomiva, Hyogo 663. Japan (K. T.
Y. S., I. U.]; Laborator9 of Chemotherapy. Aichi Cancer Center Research Institute. Chikusa-ku, Nagoya 464. Japan (T. T.. R. UI: Department of Clinical Oncology, Institute of
Development. Aging and Cancer, Tohoku University, 4-1 Seirvo-machi, Aoba-ku, Sendai 980, Japan (C. 1.1: and Department of Cancer Cvtogenetics, Research Institute for
Radiation Biology and Medicine, Hiroshima University. 1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan (M. E.. N. K.J
ABSTRACT
rangements,
Lymphoblastoid cell lines (LCLs) with heterozygous p53 mutations at
residues 286A, 133R, 282W, 132E, and 2l3ter were established from five
independent
Li-Fraumeni
syndrome
families. When cell cycle regulation
in response to ‘y-irradiation
was studied, these LCLSshowed an abnormal
G1 checkpoint associated with defective inhibition of cycin E/cyclin
dependent kinase 2 activity in all cases except for 282W LCL, which
showed a normal G1 checkpoint. On the other hand, the control of
S-phase-G2 as determined by cyclin A/cydin.dependent kinase 2 activity
was defective in all these LCLs. The mitotic checkpoint was also defective
in the two LCLs analyzed as either competent or incompetent for G1
arrest.
When
radiation-induced
apoptosis,
which
requires
wild-type
p53
function under optimal conditions, was studied, all of these LCLs showed
significant failure compared to normal LCLs. These findings indicate that
although p53-dependent transactivatlon and G,-S-phase cell cycle control
are variably dysregulated, the induction of apoptosis and control of the
cell cycle at S-phase-G2 and the mltotic checkpoint in response to DNA
damaging agents are consistently dysregulated in heterozygous mutant
LCLS. This suggests that these dysfunctions underlie, at least in part, the
susceptibility of Li-Fraumem syndrome families to cancer. Furthermore,
the approach presented is a potentially useful method for studying mdi
vidual carriers
ical features.
of different
germ-line
p53 mutations
and different
biolog
INTRODUCTION
Human cancer is a multistep process resulting from the mutation of
genes
involved
in the regulation
of cell growth,
cell differentiation,
and apoptosis. The most common target of mutation is the p53 gene,
whose product codes for a transcription factor that regulates the
expression of genes with an adjacent p53 recognition sequence, such
as the MDM2 (1), p2JciP@i/waui(2), GADD4S (3), muscle-type creat
mine kinase (4), and Box (5) genes, and leads to either cell cycle arrest
in late GI (6) or apoptosis in response to DNA damage (7). The loss
of p53 function can occur through point mutation, allelic loss, rear
by
a grant-in-aid
for
Pediatric
Research
6—5
and
a grant-in-aid
for
individual
Shimokawahigashi.
3 i'resent
Department
of
Pediatrics,
Yamanashi
Tamaho-cho, Nakakuma-gun,
address:
The
Second
Department
of
Medical
University,
1 110
Yamanashi 409-38, Japan.
Intemal
Medicine,
Nagoya
City
Univer
sity Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ka, Nagoya 467, Japan.
4 To
whom
03-3419-4757;
requests
for
reprints
should
E-mail: [email protected].
be
addressed.
Phone:
03-3414-8121;
Fax:
“hotspot―
screening
of LFS families
as well.
MATERIALSAND METHODS
EBV-transformed
LCLS. PBLs were obtained from donors and separated
by Ficoll-Hypaque gradient centrifugation. EBV-immortalized
LCLs were
established by infecting PBLs from normal individuals and symptomatic
carriers with the EBV strain B95—8as described previously (18, 19). The
were obtained
Hospital,
after obtaining
the informed
consent
of the donors
and
Tokyo.
Western and Northern
Blot Analyses.
LCLs were harvested before and 6
and 12 h after 5 Gy of y-irradiation. mRNA was purified from total RNA (70
@.tg)
using 40 @lof Dynabeads oligo(dT)25 (Nihon Dynal, Tokyo, Japan)
Japan Leukemia Research Fund; and by the Italian Association for Cancer Research.
address:
conserved
need to be established.
We report here the phenotypic abnormalities of EBV-transformed
LCLs of LFS patients with various germ-line p53 gene mutations.
Abnormalities could consistently be seen in the control of apoptosis
and in several phases of cell cycle regulation, suggesting that these
findings are significant and relevant to the susceptibility of LFS
families to genomic instability and cancer at an early stage of their
lives. We propose that the approach presented here is a potentially
useful method not only for understanding the cancer susceptibility of
these families on the basis of their germ-line p53 mutation, but for the
cancer
research from the Ministry of Health and Welfare, Japan; by a grant-in-aid from the
Ministry of Health and Welfare, Japan, as part of a comprehensive 10-year strategy for
cancer control; by a grant-in-aid from the Ministry of Education, Science, Sports and
Culture, Japan; by a grant from the Human Science Foundation, Japan; by a grant from the
2 Present
in a highly
the approval of the local committee for ethics at the National Children's
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
I Supported
deletions
family (15—17). These previous findings suggest that the ability of mt
p53 to inhibit the function of wt p53 in LFS patients may be deter
mined both genetically
and epigenetically
and that studies on the cell
biological effect of germ-line p53 mutation in individual LFS families
samples
Received I 1/13/96; accepted 3/24/97.
The costs of publication of this article were defrayed in part by the payment of page
or intragenic
region spanning the middle third of the gene. This usually leads to
disruption of specific DNA-binding and transcription, which are ap
parently critical events in the development or clonal progression of
cancer (8).
Germ-line transmission of a mt5 p53 gene in families with LFS has
revealed a new role for p53 in genetic predisposition to cancer (9, 10).
In the presence of a mutated p53, a dominant negative effect against
the wt p53 gene ( 11, 12) or a gain of function effect ( I 3) has been
identified in heterozygous cell clones. These diverse effects of p.53
mutations seem to be determined by where the mutation resides in the
p53 gene and how these mutations affect the relative expression level
of wt versus mt p53 protein (14), which may vary depending on the
5 The
abbreviations
used
are:
mt,
mutant:
wt,
wild-type;
LCL.
lymphoblastoid
cell
line;
LFS, Li-Fraumeni syndrome; PBL, peripheral blood lymphocyte; PCNA, proliferating cell
nuclear antigen; TNF, tumor necrosis factor; FASAY, functional analysis of separated
alleles in yeast; RT-PCR, reverse transcription-PCR: P1, propidium iodide: FISH, fluo
rescence in situ hydridization; TUNEL, terminal deoxynucleotidyl transferase-mediated
nick end labeling; CDK, cyclin-dependent kinase; PB, peripheral blood; MNC. mononu
clear cell.
1895
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1997 American Association for Cancer Research.
CELL CYCLE AND APOFTO5IS CONTROL IN CELLS WITH p53 MUTATION
followed by electrophoresis in 0.9% Seakem agarose gel (FMC Bioproducts,
[@y-32P]ATP.
Samples were electrophoresed in Multigel 15/25 (Daiichi Pure
Rockland,
by PCR
Chemicals).
amplification ofcDNA using the primers 5'-GCGCCATGTCAGAACCGG-3'
(forward) and 5'-CACAAACTCAGACTAAGGCA-3' (reverse) for p2ld@
irradiation)
— (kinase
irradiation)
X 100%.
waf.I
and
ME).
the
p2ld1P@h/w@@and 132MG probes
primers
were
synthesized
5'-ACCCCCACTGAAAAAGATGA-3'
5'-ATCTI'CAAACCTCCATATG-3'
(forward)
(reverse)
for (32MG.
and
Radioactivity
for
the hybridizing signals in Northern blot membrane was analyzed using a Fuji
BAS2000. Soluble total cellular protein was electrophoresed in Multigel 10
(Daiichi
Pure
Chemicals
Co.,
Ltd.,
Tokyo,
Japan)
and
transferred
to an
Immobilon polyvinylidene difluoride transfer membrane (Daiichi Pure Chem
icals Co., Ltd.). Binding of the primary antibody was detected using a com
mercial
enhanced
chemiluminescence
kit (Amersham
Japan,
Tokyo,
Japan).
The
relative
increase
activity
was
presented
without
as {(kinase
irradiation)}/(kinase
activity
activity
with
without
FISH. Culturedcells were fixed with Carnoy'sfixative solution(3:1 meth
anol and acetic acid) and spread on slides. Samples were then digested with
RNase
(100
@g/ml;Oncor)
for I h and 0.025%
pepsin
(Sigma)
for 10 mm and
fixed with formaldehyde. Samples were denatured in 70% formamide at 75°C
for 2.5 mm and hybridized with each probe at 37°Cfor 48 h in a humidified
chamber. The probes used were D1Z5, DllZl, and D16Z2 for chromosomes
1, 1 1, and 16, respectively.
formamide,
detected
The hybridized
samples
with fluorescein-conjugated
were washed
avidin-FITC,
with 50%
and counter
Antibodies to p53 (Ab2), a-tubulin, and PCNA were obtained from Oncogene
Science (Cambridge, MA). Antibodies to p2lcsP.I/war.iand CDK2 were ob
stained with P1. Hybridization signals were scored in 100 interphase nuclei
from each sample under a fluorescence microscope and a confocal laser
tamed
E and A were
scanning
Antibodies
Apoptotic Cell Determination. Flow cytometricapoptoticdeterminations
were performed as reported previously 24 and 48 h after S Gy of y-irradiation
(23). The cells were permeabilized with 0.1% saponine (Sigma) in TBS,
from PharMingen
obtained
from
(San Diego,
United
cyclin D (reactive
CA). Antibodies
Biochemical,
Inc. (Lake
with Dl and D2) and TNF-a
to cyclin
Placid,
NY).
were provided
to
by Dr. Seto (20)
and Dainippon Seiyaku Co., Ltd. Antibodies to the NH, (pAbl80l) and COOH
terminus
(HR23 1) of p53 protein
were obtained
from Oncogene
Science
and
incubated
microscope
(Olympus,
Tokyo,
Japan).
with 1 mg/ml RNase A (Sigma)
for 30 mm at 37°C, and then stained
Dr. Soussi, respectively.pAbl8Ol recognizes both wt and mt p53.
Functional Assay for Heterozygous p53 Mutations in Yeast. The FA
SAY was carriedout accordingto a methoddescribedpreviously(21). Briefly,
with 20 @g/ml
P1. After 30 mm of incubation on ice, the cells were analyzed
on a linear scale during fluorescence-activated cell-sorting analysis and quan
three centromeric
nuclear
plasmids
were constructed.
The first plasmid,
pLS76,
which
tified
for the presence
fragmented
of subdiploid,
DNA
were
also
apoptotic
detected
was used as a positive control for p53 activity, contains the LEU2-selectable
technique
marker and the human p53 cDNA coding sequences. The second plasmid,
pSSl6, is identical to pLS76 except that the central portion of p53 cDNA was
(Tokyo, Japan) in some of the samples.
replaced
by the URA3 gene.
reporter
system,
encodes
The
third
the HIS3
plasmid,
gene
p551,
expressed
which
under
contains
the
a p53-responsive
promoter. Cotransformation of the p53 cDNA PCR products, which were
amplified by PCR using Pfu polymerase (RT-PCR) so that no PCR error would
cause artifactual mutations, and p5516 cut with HindIlI and StuI results in
repair of the plasmid
with the PCR product
in vivo and constitutive
expression
of full-length human p53 protein in yeast. Clones that have repaired the
plasmid
are selected
histidine
prototrophy
on media
identifies
lacking
colonies
leucine.
Subsequent
that contain
screening
for
p53 that is transcription
ally active. Practically, 50 Leu+ colonies from each transformation, growing
on plates containing synthetic complete media lacking leucine and tryptophan,
were tested for growth on plates lacking histidine. This technique requires only
a few steps and should
mutations
permit
screening
for germ-line
or somatic
heterozygous
in the p53 gene.
Cell Cycle Analysis. LCLs were partiallysynchronizedbefore -y-irradia
tion. G0-G1cell cycle regulation was studied 16 h after S Gy of y-irradiation.
Bromodeoxyuridine
(final concentration,
10 mM) was added
to cells 30 mm
before harvesting. After fixation in 70% ethanol (—
20°C),cells were treated
with 100
@g/mlRNase
(Funakoshi,
Tokyo,
Japan).
Cells were resuspended
in
1 ml of 4N HC1and incubated for 30 mm at room temperature. Twenty @.d
of
FITC-conjugated anti-bromodeoxyuridine antibody (Becton Dickinson, San
Jose, CA) were added to cells and incubated for 30 mm at room temperature
in the dark. The cells were washed in PBS, stained for 20 mm on ice with 50
@sg/ml P1 (Sigma,
St. Louis,
MO),
and analyzed.
Ten thousand
events
were
analyzed on a FACScan cytofluorometer using the automated computer pro
gram Lysis II (Becton Dickinson). The increase (or decrease) in the percentage
of cells in the S-phase or G0-G1phase was expressed as {S-phase (or G0-G1)
with irradiation —S-phase (or G0-G1)without irradiation)/S-phase (or G0-G1)
without irradiation X 100% and analyzed statistically by the Mann-Whitney U
test.
Cells with DNA contents exceeding 4C were studied 72 and 144 h after 5
Gy of ‘y-irradiation
using P1 staining on a log scale using a FACScan or
(TUNEL)
using
a commercial
DNA.
Cells
that contained
by an in situ dUTP-labeling
kit from
Boehringer
Mannheim
RESULTS
Clinical Description. Family 1 had a p53 germ-line missense
mutation at residue 286 (GAA to GCA), as has been reported previ
ously (24). In family 2, the proband's germ-line alteration was a
missense mutation at residue 133 (ATG to AGG). She developed
unilateral and contralateral breast cancer at the ages of 26 and 27,
respectively. Her mother died of a brain tumor at the age of 43. Two
of the proband's brothers and one of her sisters developed lymphoma,
brain tumor, and bilateral breast cancer, respectively. In family 3, the
germ-line abnormality was a missense mutation at residue 282 (COG
to TGG), as reported by Shiseki et aL (25) The proband developed
osteogenic sarcoma, stromal sarcoma of the breast, and gastric carci
noma at the ages of 13, 19, and 22, respectively. Because her mother
did not show any germ-line p53 mutation, it is most likely that her
father was the affected patient carrying the germ-line mutation, al
though he could not be examined. Family 4 had a germ-line missense
mutation at residue 132 (AAG to GAG). The 15-year-old proband was
diagnosed as having rhabdomyosarcoma at the age of 2 and osteosar
coma at the age of 11. His mother and maternal grandmother both died
of breast cancer. The proband's elder brother (34 years old) had the
same germ-line p53 mutation. Family 5 was characterized by a non
sense mutation at residue 213 (CGA to TGA). The proband acquired
stomach cancer at the age of 41 and lung cancer a year later (17). The
details of families 1, 3, and 5 have been reported previously (17, 24,
25).
Expression Pattern of wt Versus mt p53 in LCLs and Precul
tured PB MNCs from Patients with a Germ-line p53 Mutation.
EBV-immortalized LCLs were established from the PBLs of symp
tomatic carriers of p53 germ-line mutations (286A, 133R, 282W,
were excluded by doublet discrimination module (Becton Dickinson).
l32E, and 2l3ter LCLs from families 1, 2, 3, 4, and 5, respectively).
Kinase Assay for Cyclin E and Cydlin A. LCLs were partially synchro
Single-strand conformational polymorphism analysis of RT-PCR
nized before y-irradiation.
LCLs were harvested 6 h after 5 Gy of ‘y-irradiation.
products showed that both the wt and mt p53 transcripts were cx
Histone Hi kinase assay was carried out according to the method described by
Dulic et a!. (22). For immunoprecipitation, 450 @gof cell extract were pressed in 286A, 133R, 282W, and 132E LCLs (data not shown). In
incubated for 60 mm at 4°Cwith 0.02 @gof rabbit polyclonal anti-human 2l3ter LCL, the expression of mt p53 was barely detectable (17).
cyclin E or A (United Biochemical, Inc.). The samples were incubated with 20 These data were confirmed by a FASAY (21), as described below.
To determine whether EBV infection might change the expression
@lof recombinant
protein A-Sepharose
beads (Zymed, San Francisco, CA) at
4°Cfor 60 mm. Immunoprecipitates were rinsed and then resuspended in 20 @l pattern of wt and mt p53 alleles, the levels of these transcripts were
of kinase buffer containing 0.5 g@gof histone HI, 2 ,.LMAlP, and 5 @Ci
of analyzed before and after EBV immortalization in three patients by
1896
FACSvantage
instrument
(Becton
Dickinson),
and the aggregation
artifacts
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1997 American Association for Cancer Research.
CELL CYCLE AND APOVFOSIS CONTROL IN CELLS WITH p53 MUTATION
PCR-single-strand conformational polymorphism. The wt:mt p53
mRNA ratio in 133R and 282W LCLs was comparable to that found
in fresh PB MNCs of the corresponding patient, even after 2 years of
in vitro culture (data not shown). Predominant expression of wt p53
was seen in 2l3ter LCL after 1 year of in vitro culture, and this was
identical to that found in precultured PB MNCs, as reported previ
ously (17).
A
1
2
3
4
6
5
—
HRS:0612
@
0612
0612
0 612
0612
-@-
--I-
32.7Kd
J@t
p21
0 612r
TransactivationActivityof p53 ProteinEncodedby Separated
17.7Kd
Alleles in LFS-derived LCLS. To clarify the functional activity of
these mutations, we used an assay based on the FASAY, which allows
the determination of the transactivating property of each mt species of
p53 under homozygous conditions. As a result, 286A, 133R, 282W,
and 132E LCLs were found to have 48, 41, 40, and 29% His+
colonies, respectively, among the total colonies analyzed (Table 1),
indicating that these mutations are transcriptionally inactive when
homozygous. This result also indicates that approximately 30—50%of
p53 transcripts were derived from the mt p53 allele in these LCLs.
These findings were in contrast to the 98—100% His+ colonies
observed in normal controls and the 88% His+ colonies observed in
2l3ter LCL. The level seen in the latter is consistent with the finding
that the expression of wt p53 predominated over that of mt p53 in
2l3ter LCL (17).
@
@
@
@
@
@
@
Radiation-induced
p2lciPI/wafi
transactivation
is mediated
by TNF-a
in some leukemic cells (27). To determine whether this cytokine was
involved in our system, p2lciPI/waui induction was examined in
irradiated LCLs cultured in the presence or absence of excess amounts
of neutralizing anti-TNF-a antibody. However, no decrease in p2lC@
transactivation
anti-TNF-a
was
found
in
normal
LCLs
exposed
to
the
antibody. This rules out the possibility that this cytokine
Table
LCLSThe
1 Functional assay of mt p.53for separated allele in yeast in LFS-derived
+colonies
function of each mt p53 allele was analyzed as described in the text. Leu
syntheticcomplete
from each transformation (total colonies) growing on plates containing
lackinghistidine
media lacking leucine and tryptophan were tested for growth on plates
@
(His+).His@/total
coloniesLCL
(%)wtlwt
analyzed
@—
—
— —
- a-tubulin
B
1
HRS: 06
2
3
4
06
06
06
‘1
•@1P21
Induction Is Variably Dysregulatedin LFS-de
rived LCLs. @21ci@-l/waf1 is the major inhibitor of the activities of
cyclin/CDK/PCNA complexes and is transactivated by p53 in re
sponse to DNA-damaging agents. Accordingly, we studied the p53induced expression of p21Ci@@5a@6 and 12 h after S Gy of -y-irra
diation in these LFS-derived LCLs.
protein was found to
be barely detectable in any of the untreated LCLs. After irradiation, a
remarkable increase in p2lciPl/wafl protein was seen in normal LCLs.
On the other hand, the induction of @21ciPl/wafiprotein in irradiated
286A, 133R, and 132E LCLS remained significantly low; levels were
less than 50% of that seen in irradiated normal LCL throughout the
12-h observation period. In 282W and 2l3ter LCLs, p2lciPl/waf 1
protein was transactivated by up to 80 and 50%, respectively, of that
of the irradiated normal LCL (Fig. 1A). These findings were supported
by Northern blotting of 286A, l33R, and 282W LCLs 6 h after
irradiation. @21ci@-I/waf-I
mRNA increased markedly in normal LCLs.
Although 282W LCL showed slightly less enhanced expression, it
was still significantly higher than that seen in 286A and 133R LCLs
(Fig. 1B). The leukemic cell lines HL-60 and KOPM28 (26), with null
or mutated p53 expression, respectively, showed no expression of the
p2l―@1/waf-I transcript irrespective of irradiation (data not shown).
I/waf-I
—
.
N
@@2-MG
Fig. I. p21C@@
l/@af@
I expression after irradiation in LFS-derived LCLS with a heterozy
gous p53 mutation. A. western blotting for
protein transactivated before (0),
6 h (6), and 12 h (12) after irradiation.
Lane 1, wt/wt control;
Lanes 2—6, 286A,
282W, l32E, and 2l3ter LCLS, respectively. B, Northern blotting for p2lc@l
133R,
mRNA
transactivated before (0) and 6 h (6) after irradiation. Lane 1, wi/wI control; Lanes 2—4,
286A, 133R, and 282W LCLs, respectively.
is involved in our LCLs and is consistent with the results observed in
other leukemic cell lines (Refs. 27 and 28; data not shown). These
results indicate that the level of p2l―@1/W5f1 induction after DNA
damage is impaired to a variable degree, depending on the nature of
the p53 mutation in the LFS-derived LCLs.
Cell CycleDysregulationin LFS-derivedLCLsin Responseto
y-Irradiation.
It is well known that cell cycle arrest in G1 in response
to DNA damage is mediated by p53-induced
transacti
vation. To assess p53 function, 0 growth arrest induction was cx
amined in these LCLs 16 h after S Gy of -y-irradiation. The LCLs with
286A, 133R, 132E, and 2l3ter mutations showed a slowdown of
I arrestinduction
(thepercentage
of increase
in
1 was
—3.9±7.3, —6.6±7.5, —4.0±0.29, and 1.2 ±6.1%, respectively;
Fig. 2). In contrast,
282W
LCL showed
a significant
increase
in the
G0-G fraction (percentage of increase, 7.4 ±10.0%) after irradiation
up to a level that was not significantly different from that seen in
normal LCL (percentage of increase, 10.5 ±7.6%). To confirm this
unexpected feature of the 282W mutation, another clone, 282W LCL',
was established from the elder sister of the proband in family 3. 282W
LCL' also showed a significant increase in the G@-G@fraction after
irradiation
(data not shown).
This supports
our finding
that the 282W
mutation is unique among the heterozygous mutations with regard to
cell cycle control. The abnormalities in 0 cell cycle regulation in
286A, 133R, I32E, and 2 13ter LCLs are consistent with defective
p2ld1@wo@i
induction
in these
LCLs,
which
most
likely
results
from
the dominant negative transactivation activity of mt pS3 species when
98-100286A
48133R
41282W
40l32E
292l3ter
heterozygous.
88
Abnormal Inhibition of Cyclin E- and A-associated Kinase
Activity
in LFS-derived
LCLS after
@-Irradiatlon. Cyclin
EJCDK2 and cyclin A/CDK2 complexes have been shown to be
I897
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1997 American Association for Cancer Research.
CELL CYCLE AND APOFFOSIS CONTROL IN CELLS WITH p53 MUTATION
S phase
GOIG1 phase
p < 0.05
p@
U
0.5175
_.101
p<0.05
I
I
p<ft05
Up.cO.06
%I
1
I
p=0.0e
I
% I
20@
30
10@
p<0.05
I D(0.05
15
0
I
—1(
p <0.05
p<0.05
p <0.05
No.1
No.2 No.3
No.4
No.5 No.6 No.7
No.1
No.2
No.3
No.4
No.5 No.6 No.7
Fig. 2. G0-G1 cell cycle regulation in LFS-derived LCLs after ‘y-irradiation.The statistical analysis of 10 independent experiments in LCLS is shown. The increase (or decrease)
in the percentage of cells in the S-phase or G0-G1 phase was expressed as (S-phase (or G0-G,) with irradiation —S-phase (or G0-G1) without irradiation}/S-phase (or G0-G1) without
irradiation X 100% and analyzed statistically by using the Mann-Whitney U test. Nos. 1—6,wt/wt, 286A, 133R, 282W, 132E, and 2l3ter LCLs, respectively. No. 7, leukemia cell line
HL-60 with no p53 expression. Left panel, percentage of increase in cell population in the G0-G1 phase after y-irradiation compared to that without irradiation. Right panel, percentage
of increase in cell population in S-phase after ‘y-irradiationcompared to that without irradiation.
essential for the G@to S-phase and S-phase to 02 transition, respec
tively, and both of them are subject to inhibition by p2ldi@hI'@I in
response to DNA-damaging agents. As reported above, 286A, 133R,
132E, and 2l3ter LCLs but not 282W LCL showed dysregulation of
the G1-S-phase checkpoint associated with defective p21ciP.i/waui
transactivation after y-irradiation. These observations prompted us to
investigate the histone Hi kinase activity associated with cyclinlCDK
complexes (Fig. 3). Inhibition of cyclin EJCDK2-associated histone
HI kinase activity was significantly reduced in 286A, l33R, l32E,
and 2 13ter LCLs, but not in 282W LCL (Fig. 3A). The substantial
inhibition
of cyclin
E-associated
kinase
consistent with normal radiation-induced
inhibition
of cyclin
A-associated
histone
activity
in 282W
LCL was
LCLs. Cellsfrommicedeficientin wt p53 displaya reducedsensi
G@arrest. In contrast, the
HI kinase
activity
was not
sufficient in any of the LFS-derived LCLs, including 282W LCL (Fig.
3B),suggestingthatcontrolof S-phase-G2
isdisruptedintheseLCLs.
@
population started to accumulate at 72 h and continued to increase
until 144 h after irradiation. There was a notable difference in the
proportion of octaploid cells between normal LCL (less than 1.0%)
and LCLs carrying the 286A (1 1.0%) or 282W (9.0%) mutation at
144 h after irradiation (Table 3). These findings were also confirmed
by FISH using centromere probes for chromosomes 1, 11, and 16 (Fig.
4B). These results indicate that LFS-derived LCLs consistently show
significant defects in the mitotic/spindle checkpoint and that they are
highly susceptible to genomic instability in response to a genotoxic
substance.
Radiation-induced
Cell Death Is Dysregulated in LFS-derived
To rule out the possibility that the abnormal cell cycle kinetics and the
change in the biochemical characteristics of cyclin/CDK complexes
only reflected the level of cyclin E or A, the expression of these
cyclins was analyzed by Western blotting. No significant difference in
the expression of these cyclins or in that of CDK4 and PCNA was
found in LCLs carrying either the wt/wt or wt/mt p53 when the cells
were examined 16 h after ‘y-irradiation(data not shown).
tivity to DNA-damaging agents and an increased failure to enter
apoptosis (29). To determine whether p53 heterozygous mutations
confer resistance against cell death stimuli, LFS-derived LCLs were
irradiated with 5 Gy and examined for apoptotic cells 24 and 48 h
later. At 24 h, the percentage of the subdiploid fraction was slightly
greater in normal than in LFS-derived LCLs, and this difference was
significantly increased at 48 h, with 28.63 ±3.28% in normal LCL
and 9.56, 9.47, 13.62, 5.17, and 7.20% in 286A, l33R, 282W, 132E,
and 2 l3ter LCLs, respectively (Table 2). 282W LCL, despite exhib
iting an apparently
normal
arrest,
was nonetheless
modestly
Defect in Control of the Mitotic Checkpointin LFS-derived resistant to radiation-induced
apoptosis (Table 2 and Fig. 4). One of
LCLs. p53hasbeenimplicatednotonlyin a G@checkpoint,butalso the data in normal, 286A, and 282W LCLs 48 h after irradiation was
in the later phases of the cell cycle, such as PCNA-dependent DNA
replication and the mitotic checkpoint, both of which are essential for
the maintenance of diploidy. To investigate a possible abnormality in
the mitotic checkpoint in LFS-derived LCLs, in 286A and 282W
LCLs, which showed 20 and 80% of the p2Id1P@h/5@1@
transactivation
of a normal LCL, respectively, the proportion of the cells that devel
oped a second round of DNA synthesis without cytokinesis after 72
and 144 h of exposure to S Gy of y-irradiation was examined.
Characteristic phenotypes such as decreased apoptosis (shown later),
moderate G2 delay, and accumulation of DNA contents greater than
4C were seen in both of the heterozygous LCLs (Fig. 4A). The 8C cell
further verified by the TUNEL method, and 53, 16.3, and 13.2% of the
cells were shown to be in the apoptotic population, respectively (data
not shown). All these data suggest that the induction of apoptosis is
consistently
mutation.
impaired
in LCLs
with various
types
of germ-line
p53
DISCUSSION
To study the biological behaviors of LFS-derived LCLs heterozy
gous for p53 gene mutations, control of the cell cycle and apoptotic
cell death in response to y-irradiation were investigated. LCLs were
1898
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CELL CYCLE AND APOPrOSIS CONTROL IN CELLS WITH p53 MUTATION
A
IR (-) (-i-)
%
1
20'
2
10@
{(kinase activity with irradiation) —(kinase activity
activity without irradi
7
7
— —
-50
B
%
IR@
30
1
2
3
4
5
6
7
20
10
0
-10
-20'
-30'
-40
-50
-60
3—
4
5
6
.
7
@
6
-40
2@
@
5
-30
6@
results of three independent experiments is shown.
Lanes 1—6,wtlwt, 286A, 133R, 282W, l32E, and
without irradiation)}/(kinase
ation)X 100%.
4
-20
5
cording to the method described in the text, using
histone Hi as a substrate. One of the representative
tometric value of phosphorylated histone Hi after
‘y-irradiation.The relative increase was presented as
3
-10
4@
2l3ter LCLs, respectively. Lane 7. leukemia cell
line HL-60 with no p53 expression. Leftpanel, blots
of phosphorylated histone HI. Right panel, ache
marie diagram of the relative increase in the densi
2
0
3
Fig. 3. Cyclin E- and cyclin A-associated kinase
activities in LFS-derived LCLs after ‘y-irmdiation.
LCLS were partially synchronized and incubated for
6 h after 5 Gy of ‘y-irradiation.
Cyclin E (A)- and A
(B)-associated kinase activities were measured ac
1
established from symptomatic carriers representative of the five LFS
families showing different constitutional p53 mutations. EBV-immor
talized LCLs maintain expression of both wt and mt p53 alleles during
long-term in vitro culture, as demonstrated by RT-PCR and FASAY
(in this paper and Ref. 18), possibly due to partial transformation by
EBV-encoded EBV nuclear antigens and latent membrane protein-l.
Although EBV nuclear antigen 5 and BZLF1 have been reported to
bind to p53 protein (30, 31), this interaction seems to be negligible
with regard to p53-dependent transactivation and cell cycle control, at
least in in vitro transformed LCLs (32). We and others have seen that
the relative level of wt versus mt p53 mRNA varies among families
with germ-line p.53 gene mutations (15—17).The expression pattern of
wt versus mt p53 in precultured PB MNCs has been preserved in
LCLs from families 2, 3, and 5 (data not shown). These findings
indicate that we can investigate the functional properties of each mt
p53 in a heterozygous context using EBV-immortalized LCLs.
p53 is a tumor suppressor gene with pleiotropic functions that
include the control of genomic plasticity and integrity. Transactivation
of p21ciPI/W@fl by p53 seems to be indispensable in these processes.
Although the function of p2l@―@@ has not been fully elucidated,
inhibition of the kinase activity of multiple cyclin/CDK complexes
has been shown
to play a central
role in cell cycle
regulation.
DNA
damage-induced inhibition of cyclin E-associated kinase activity was
absent in 286A, l33R, l32E, and 2l3ter LCLs, but not in 282W LCL.
These findings are consistent with the results of cell cycle analyses
showing
that 286A,
l33R,
l32E,
and 2l3ter
LCLs fail to arrest in GI
after irradiation, whereas 282W LCL was significantly blocked in G@.
On the other hand, the inhibition of cyclin A-associated kinase activity
was insufficient
in all of the heterozygous
clones,
including
Table 2 Increase and48
in the apoptotic cell population before irradiation
LCLSThe
h after 5 Gy of irradiation in LFS-derived
282W
(0 h) and 24
Thevalues
apoptotic cell population was measured by FACScan as described in the text.
genotype).LCL
are the means ±SD (n > 3 for each
48hwt/wt
Oh
24h
3.28286A
2.07 ±0.76
11.98 ±4.96
1.37―l33R
2.68 ±1.85
5.52 ±1.53―
1.90―282W
3.99―132E
2.15―2l3ter
2.92
2.63
1.85
2.67
5.49
5.03
254
5.16
±1.24
±1.89
±1.68
±1.31
±159b
±3.05―
±1.30―
±2.28―
28.63 ±
9.56 ±
13.62 ±
5.17 ±
7.20 ±5.35―
“P< 0.05.
b ,,
o.os, Mann-Whitney U test.
1899
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CELL CYCLE AND APOPTOSIS CONTROL IN CELLS WITh p53 MUTATION
A
wt/wt LCL
286A LCL
50(
50(
C,)
(‘I.@
VII
0
0
U
:[@
I
101
@
I
[email protected]@
50(
2C$$
Cl,
Cl)
C
C
0
C.)
0
I
I
I
1O@ iø@ i04
101
i@3
i@:l@
101
I
L
1O@lO@ iO'@
FL2-Height
FL2-Height
5O@
2C @4C@L
0
FL2-Height
72h
282W LCL
500
Cl)
Cl)
C
C
8eA@@Ai@.
0
0
I
101
T
I.
I
101 i@2 io@' io@
iø2 iø@ i04
FL2-Height
FL2-Height
FL2-Height
FL2-Height
FL2-Height
@r'l
144h
@&-.
I
Ii
I
101
j@2
L
io@
io@
FL2-Height
B
.
S
.
*1
Fig. 4. A, flow cytometric analysis of the mitotic checkpoint in LFS-derived LCLS. LCLs before (Oh), 72 h (72h), and 144 h (144h) after irradiation were analyzed with regard to
their DNA contents by flow cytometry as described in “Materialsand Methods.―Two heterozygous LCLs (286A and 282W) with different transactivation activity of @21cIP@I/waf@l
and
wtlwt LCL are shown. B, FISH signals in a polyploid cell seen in 286A LCL 144 h after ‘y-irradiation.
FISH signals are seen as white dots, and nuclear morphology is revealed by
P1 staining. FISH analysis showed 20% of the treated cells contained more than 4 copies of chromosome 1, 11, and 16. An example using DIZ5 probe for chromosome 1 is shown.
@
LCL. On the basis of these findings, all of the LFS-derived LCLs
seem to have a defect in the control of S-phase-G2 in response to
DNA-damaging agents, suggesting that this defect may universally
underlie the susceptibility of LFS patients to cancer. These findings,
along with the p2lC1P@'1@@@
transactivation data, also suggest that
although inhibition of cyclin E/CDK2 kinase can be achieved by a
certain threshold concentration of p2lCiP 1/waf-I, inhibition of cyclin
A/CDK2-associated
kinase activity may require an even higher
threshold level
. These interpretations are supported by
the previous observation that cyclin E/CDK2 has a higher affinity for
1900
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CELL CYCLE AND APOPTOSI5 CONTROL IN CELLS WITh p53 MUTATION
Table 3 Sum,nary of the characteristics
of LCLS with various germ-line p53 mutations
Wt p53 mRNA percentage was determined by FASAY.
i
G@JG1―
cells―wt/wt
LCL
wt p53 mRNA (%)
arrest
98-100
Cyclin AApoptosis―Octaploid
p21 transactivation―Kinase Cyclin Enhibition―
Normal
Normal
286A
48
Defective
<50%b
133R
41
Defective
<50%―
282W
40
Normal
29
88
Defective
Defective
132E
NTa
2l3ter
80%―
<50%b
50%―Yes
Analyzed
on
cells
with
orwithout
y-irradiation,
as
described
inthe
text.
@
b
C NT,
@
@
@
@
No
No
No
No
11.0%
NT'
Yes
No
No
9.0%
No
NoYes
No
NoYes
No
No<1%
NT
response as compared to normal LCLS.
not
tested.
binding (33). Thus, differences in the effective threshold
concentration of p2lciPl/waf4 after irradiation may explain the phe
notypic diversities of cell cycle control in these LCLs.
The apparently insufficient transactivation of p2lC@Pl/waf 1 suggests
that the defect in cell cycle control in LFS-derived LCLs is not limited
to G1 or S-phase-G2 phase. The nütoticcheckpoint has also been
shown to be under the control of p53 (34). This finding was further
extended by Waldman et a!. (35), who showed that S-phase-M-phase
coupling in response to a DNA-damaging agent is controlled by
Two representative LFS-derived LCLs, 286A and
282W, with minimum and maximum
transactivation
activity were studied with respect to the proportion of cells with DNA
contents greater than 4C. The results showed that both of these LCLs
were associated with a significant accumulation of polyploid cells
with DNA contents exceeding 4C. This indicates that the deregulation
of the mitotic/spindle checkpoint is a common biological abnormality
in LFS-derived LCLs. Thus, in the presence of a DNA-damaging
agent, there seems to be a variable abrogation of G1 arrest and
consistent dysregulation of the S-phase-G2 phase and mitotic check
point in LFS-derived LCLS.
In all of these LCLs, a defect in the induction of apoptosis in
response to y-irradiation was seen by P1 staining, part of which was
further verified by the TUNEL method. In the light of results showing
that
mice possess a normal response with re
spect to apoptosis (36), the defect that we see in apoptosis is difficult
to account for on the basis of a poor response in
transactivation. Rather, these findings support the hypothesis that
p53-dependent activation for apoptosis requires different target genes
(37). Alternatively, the inhibition of apoptosis could be directly in
duced by the mt allele of the p.53 gene in the form of a gain of function
effect in 286A, 133R, 282W, and 132E LCLs or in a manner that
relies on the level of wt p53 function in 2l3ter LCL. The expression
of proteins of the bcl-2 gene family was analyzed by Western blotting
to determine their relevance to the observed failure of LCLs to enter
apoptosis. Bcl-2 and bax proteins are of particular interest, because
their transcription is reciprocally regulated in cells undergoing apop
tosis, apparently by the interaction of p53 with regulatory DNA
sequences present in their genes (5). The steady-state levels of bcl-2
and bax proteins, however, remained unchanged during the 48-h
period after irradiation in these clones, including normal LCLS,6
suggesting that these genes do not directly mediate apoptosis in our
cells. It should be noted, however, that p53 may trigger apoptosis in
the absence of transcriptional activation of p53 target genes (38). In
LFS-derived LCLS, 282W LCL showed a less significant reduction of
the transactivation activity of p21C1Pl/W@f@l@
A FASAY enabled us to
show that the 282W mutation has no transactivation activity, as in the
case of other mutations. This is consistent with the well-established
6 D.
No
No
Delia
and
S. Mizutani,
unpublished
observation that the 282W mutation abrogates the sequence-specific
DNA binding capacity (39, 40). These findings may suggest that mt
pS3 species,
which
are inactive
with regard
to transactivation
in a
homopolymerized context, can variably interfere with wt p53 function
in a heterozygous context. Our findings point to the need for a study
of the biological features of cells from carriers with individual germ
line p53 mutations.
The results presented here argue against a recent report showing
that LCLs with 257Q, 342ter, and 257FS heterozygous mutations
maintain p53 function during long-term in vitro culture (18). Although
their study did not include an analysis of the mitotic checkpoint, it is
likely that the apparently normal characteristics of those LCLs corre
spond to those seen in 282W LCLs, suggesting that these mutations
will also show mt p53 activities in the control of the later phases of the
cell cycle and apoptosis when analyzed in the presence of a DNA
damaging agent.
In summary, although the impairment of G1 cell cycle control is
variable, dysfunctions in the control of apoptosis and cell cycle
control at S-phase-G2 and the mitotic checkpoint are consistently seen
in LFS-derived
LCLs
in response
to DNA-damaging
agents,
thus
suggesting that these dysfunctions underlie, at least in part, the sus
ceptibility of LFS families to cancer. Furthermore, the approach
presented here is a potentially useful method for studying individual
carriers of different germ-line p53 mutations and different biological
features.
ACKNOWLEDGMENTS
We are grateful to Dr. Seto of the Aichi Cancer Center, Dr. Kitaura of
Dainippon
Seiyaku
Co.,
Ltd.
and Dr. Soussi
of the Institut
Dc Genetique
Moleculaire for their generous gifts of anti-cyclin D, anti-TNF-a, and anti-p53
antibodies (HR231), respectively. We thank Dr. Miyashita for a critical reading
of this manuscript.
We thank M. Komai
for her technical
assistance.
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DNA Damage-associated Dysregulation of the Cell Cycle and
Apoptosis Control in Cells with Germ-line p53 Mutation
Kumiko Goi, Masatoshi Takagi, Satoshi Iwata, et al.
Cancer Res 1997;57:1895-1902.
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