[CANCERRESEARCH54. 3718—3722.
July 15. 19941
Advances in Brief
Cell Cycle Arrests and Radiosensitivity of Human Tumor Cell Lines:
Dependence on Wild-Type p53 for Radiosensitivity'
Amanda J. Mcllwrath, Paul A. Vasey, Gillian M. Ross, and Robert Brown2
CRC Department of Medical Oncology, CRC Beatson Laboratories, Switchback Roa4 Garscube Estate, Bearsden Glasgow
Radiotherapy Research Unit, Institute of Cancer Research, Sutton 5M2 5NG fG. M. R.J, United Kingdom
Abstract
Loss of p53 function
has been shown to cause increased
resistance
to
ionizing radiation in normal murine cells; however, the role of p53 in
radloresistance
of human
tumor
cells
is less clear.
Since
wild-type
p53
function is required for radiation-induced G1 arrest, we measured G1
arrest in 12 human tumor cell lines that have a wide range of radiosen
sitivities (surviving
fraction at 2 Gy, 0.11—0.8). We observed
a significant
correlation between the level of ionizing radiation-induced G1 arrest and
radiosensitivity. Cell lines having G1 arrest are more radiosensitive. There
is no correlation between maximal G2 arrest and radiosensitivity. Expres.
sion of a dominant-negative mutant of p53 (codon 143, Val to Ala) in
transfectants of the radiosensitive human ovarian cell line A2780 abro
gates
the radiation-induced
G1 arrest.
Such
mutant
p53 transfectants
p53 is transfected into a human colon tumor cell line, such that a
radiation-induced p53-dependent G1 arrest is abrogated, the cells do
not change their radiosensitivity (1 1). The lack of effect on radiosen
sitivity in this line may be due to expression of other resistance
mechanisms which overwhelm any impact on sensitivity of modulat
ing p53 function. We have used a similar approach to investigate the
effect on radiation sensitivity of losing the radiation-induced G1 arrest
in a radiosensitive human ovarian cell line.
Materials
p53 function
Is required
for sensitivity
and Methods
Cell Culture, Radiation Treatment, and Flow Cytometry. The cell lines
used in the present study are shown in Table 1. All cell lines were maintained
are
as monolayers in RPMI 1640 with 10% fetal calf serum and grown at 37°Cin
more resistant to ionizing radiation than the parental line and vector
alone transfectants, as measured by clonogenic assays. These results sup
port the concept that wild-type
95% air/5% CO2. Exponentially
growing
cells were irradiated with y-rays at
room temperature using a @‘°Co
source delivering 2Gy/min. DNA synthesis was
of
assessed by incorporation of BrdUrd3 and flow cytometric analysis as de
scribed previously (1, 9). Exponentially growing cells were @yirradiated, at
tumor cells to DNA-damaging agents, such as ionizing radiation, and that
the loss of p53 function in certain human tumor cells can lead to resistance
to ionizing radiation.
various times medium containing 10 @iM
BrdUrd was added, and the cells were
incubated for 4 h at 37°C.The cells were harvested and fixed; after denatur
ation of the DNA
Introduction
@
G61 JBD fA. J. M., P. A. V., R. B.J, and
mouse
with 2 N HC1, cells were incubated
MAt, (Dako).
Bound complexes
were detected
with an anti-BrdUrd
with goat anti-mouse
fluorescein isothiocyanate (Sigma, Poole, United Kingdom), stained overnight
Damage to the DNA of proliferating cells by ionizing radiation
causes an arrest of mammalian cells in the G1 and G2 phases of the
cell cycle (1 , 2). Absence of such cell cycle arrests has been shown to
be associated with hypersensitivity of eukaryotic cells to ionizing
radiation (1—3).Certain types of tumor, such as neuroblastomas and
testicular germ cell tumors, are exquisitely sensitive to radiotherapy as
well as chemotherapy, in comparison to the majority of tumors (4).
Furthermore, cells derived from such tumors retain a sensitive phe
notype in vitro (5, 6). This leads to the hypothesis that radiosensitive
tumors may have genetic defects in cell cycle arrest equivalent to
those present in radiation-sensitive mutants of yeast or mammalian
cells (1, 7). In the present study, we have analyzed G1 and G2 cell
cycle arrests induced by ionizing radiation in human tumor cell lines
with a wide range of radiosensitivities. We show that G@arrest but not
G2 arrest correlates with radiosensitivity. Since radiation-induced G1
arrest in mammalian cells has been shown to require wild-type p.5.3
expression (1), we have examined expression of p53 in more detail in
these lines. Lack of G1 arrest and loss of p53 function has been
correlated with acquired or intrinsic resistance of cells to DNA
damaging agents such as ionizing radiation and cisplatin (8, 9). Since
wild-type p53 has been shown to be required for ionizing radiation
induced cell death by apoptosis (10), this may explain why loss of p53
function as measured by loss of G1 arrest correlates with resistance.
However, it has been shown that when a dominant-negative mutant of
at 4°Cwith propidium iodide, and analyzed by flow cytometry using a Coulter
(Hialeah, FL) EPICS Profile Analyzer.
Immunoassays. Cell extracts were prepared by lysing exponentially grow
ing cells in 1% Nonidet P-40, 500 m@iNaCI, 50 mM Tris (jH 7.5), and I mM
dithiothreitol in the presence of protease inhibitors. Protein concentrations
were determined by the Bio-Rad (Richmond, CA) protein assay. Immuno
blotting was carried out as described previously (9) using the p53 antibody,
pAB2
(Oncogene
Sciences,
chemiluminescence;
Manhasset,
NY)
and
visualized
the intensity of the autoradiographic
by enhanced
signal was quantified
by laser densitometry. For EUSA analysis, a sandwich immunoassay was used
to measure p53 levels in cell extracts as described previously (12) with
anti-p53
MAb
240
(13)
as the solid-phase
reagent.
Levels
of p53
were
quantitated by reference to a calibration curve using purified recombinant p53.
DNA Transfections and Drug Sensitivity Assays The plasmid pCS3SCX3 (14) containingmutantp53 complementaryDNA (codon 143, Val to
Ala) expressed from a CMV promoter and vector alone without insert were
transfected into the cell line A2780 (9). Expression of p53 in individual clones
was assayed by ELISA as described above. For clonogenic drug sensitivity
assays,
cells were seeded into 10-cm2 plates and 24 h later were irradiated
with -y-rays from a @°Co
source. After incubation of the plates for 10 days,
colonies were stained, and those greater than 200 cells were counted.
Results
Radiation-induced
Cell Cycle Arrests. Table 1 summarizes the
cell lines used in the present study. The sensitivities of the cell lines
to ionizing radiation have been determined by cbonogenic assays.
Shown in Table 1 are the respective surviving fraction of clonogenic
cells after 2 Gy irradiation (SF2Gy). Cell lines that were analyzed
Received 4/26/94; accepted 6/1/94.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
‘This
work was supported by the Cancer Research Campaign (United Kingdom) and
the Scottish Hospitals Endowment Research Trust.
2 To
whom
requests
for
reprints
should
be addressed.
3 The abbreviations
used are: BrdUrd,
body; ELISA, enzyme-linked
2 Gy.
bromodeoxyuridine;
immunosorbent
MAb,
monoclonal
anti
assay; SF2Gy, the surviving fraction at
3718
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CELL CYCLE ARREST AND RADIOSENS@VITY
Table 1 Radiosensitivity and cellcycle
arrestCell
240g
2 GyG2― ESVFold
Gy%5CESF―%
ELISARadiosensitive
lineOrigin―SF2Gyb2
HX142
1
NB1
Neuroblastoma
SUSA
Teratoma
23
p531MAb
2
Neuroblastoma
0.15
0.16
0.21
45
43
24
35
53
24
228
476
153
319
345
153
3.2
2.1
6.8
1.5
10.4
1.6
Ovarian Adeno.
Teratoma0.1
0.22
0.2710
23
4126
23
41145
259
259
1881
4.5
4.2
2.8
U251
Glioma
0.56105562914631.012.40.5779140275316ND―o0.58162741352921.4170.6291721652861.2350.6694871331591.5630.771
MGH-U1
Bladder TCC
MOG-G-CCM
RT112
MOG-G-UV
IP-SB18
Glioma
BladderTCC
Glioma
Glioma
SK-N-SH
A2780
GCF27Neuroblastoma
1881
2.04
Radioresistant
@
a Type
of
tumor
from
Glioma
which
cell
line
was
derived.
Adeno.,
adenocarcinoma.
b The surviving fraction of clonogenic cells after 2 Gy irradiation.
C Percentage
of
cells
incorporating
BrdUrd
24
h
after
irradiation
d Percentage of maximal increase in cells with 4N DNA
compared
to
untreated
cells.
content after irradiation compared to untreated cells.
e Cells irradiatedto give approximately equivalent clonogenic surviving fraction (ESF) of 0.2—0.3.
1The maximumfold increasein p53proteinafter2 Gy irradiationcomparedto untreatedcells.Levelsof p53 weredetectedby Westernimmunoblotting
usingMAb 1801and
quantitated by laser densitometry.
g
@53 protein
(ng/mg)
cell
extract
detected
by
ELISA
using
MAb
240
which
recognizes
a p53
epitope
exposed
when
p53
is
in
mutant
conformation
(13).
The
levels
of
p53
were
quantitated by comparison with a calibration curve using recombinant p53 and using a concentration of cell extract which is in the linear range of the ELISA.
h ND, p53 protein not detected.
were categorized as radiation sensitive if they had a SF2Gy value of
less than 0.35. Conversely, radioresistant lines were defined as having
a SF2Gy value of greater than 0.35. The radiosensitive lines (SF2Gy,
0. 11—0.27)were derived from neuroblastoma, testicular and ovarian
carcinoma, tumor types that can be highly responsive to radiotherapy
in vivo (4, 5). The radioresistant lines (SF2Gy, 0.56—0.8)were de
rived from glioma and bladder carcinoma, tumors that respond poorly
or relapse rapidly after radiotherapy (5, 6).
At various times after -y-irradiation, the percentage of cells in S
(BrdUrd positive) and at G2/M (4N DNA content) of the cell cycle has
been measured compared to control, untreated cells. The maximal
decrease in the percentage of S cells was observed 24 h after irradi
ation. All of the radiosensitive lines showed markedly more inhibition
of DNA synthesis after irradiation with 2 Gy than the radioresistant lines
(Table 1). The radiosensitive cell lines showed more than 50% maximal
inhibition of DNA synthesis after irradiation with 2 Gy, whereas the
radioresistant lines showed little or no inhibition of replicative DNA
synthesis.
The level of inhibition
of DNA synthesis
significantly
corre
bated with SF2Gy, using a Pearson's correlation (r = 0.808; P < 0.01;
Fig. IA). As well as comparing the cells using equidoses of irradiation,
the cell lines were compared using doses of irradiation that gave approx
imately equivalent surviving fractions of 0.2—03(Table 1). Significant
correlation is observed between SF2Gy and the percentage of S cells at
24 h after irradiation using a dose of radiation that gave equivalent
survival (r = 0.795; P < 0.01; Fig. 1B). These results show that the
radiosensitive cell lines all have marked G1 arrest after irradiation,
whereas the radioresistant lines have little or no G1 arrest.
All of the lines show G2 arrest as measured by the increase in cell
number with a 4N DNA content after irradiation (Table 1). Although
a range in the maximal level of G2 arrest observed for each line is
observed after irradiation (35 to 376% increase in G2 cells after
irradiation compared to untreated cultures), there was no significant
correlation between SF2Gy values and the percentage level of G2
arrest observed using either 2 Gy or equivalent survival doses (Fig. 1,
C and D). Therefore, the presence of G1 arrest but not G2 arrest
appears to correlate with the radiosensitivity of these lines. There was
also no significant correlation between the level of G2 arrest and the
percentage reduction in S cells, implying that the G1 arrest observed
in the radiosensitive lines is not due to inhibition of cell cycle
progression deriving from the G2 arrest.
Accumulation of p53 in Radiosensitiveand Radioresistant
Lines. As shown in Fig. 2, all of the radiosensitive lines had increased
levels of p53 protein 8 h after irradiation with 2 Gy y-rays. On the
other hand, the radioresistant lines showed little significant difference
in levels of p53 protein after irradiation at any of the time points
examined (Table 1). However, all of the radioresistant lines showed
markedly higher levels of p53 protein than the sensitive lines, with the
exception of the MGH-U1 cell line, which has no detectable p53
protein before or after irradiation. The high levels of p53 observed in
the radioresistant lines may be indicative of these lines containing
nonfunctional p53 protein, which has a longer half-life due to mis
sense mutation in the p53 protein or stabilization due to the binding to
other cellular proteins (15). Many of the missense mutations that
increase the p53 half-life also cause a conformational change in the
p53 protein,
which
can be detected
by cross-reaction
with p53 anti
bodies such as MAb 240 (13). The ability of MAb 240 to cross-react
with p53 from the radiosensitive and radioresistant lines has been
quantified by ELISA (Table 1).
All of the radioresistant
lines show high levels of expression
of p53
as quantified by ELISA using MAb24O, except for the line MGH-U1
which had undetectable levels. Two of these lines, T98G and U251,
are known to express missense mutants of p.5.3 (16). The radiosensi
tive lines all express p53 that can be detected by ELISA, in particular,
the SuSa cell line, but all of these sensitive lines have less p53 protein
in a mutant conformation, as detected by cross-reaction with
MAb24O, than the radioresistant lines. Two of the radiosensitive lines
(A2780 and SK-N-SH) are known to only express wild-type p53 (9,
17). The cross-reaction
detected
in the radiosensitive
cell lines appears
to be p53-specific since HL-60 cells, which have the p53 gene deleted
(18), show no cross-reaction, and A2780 cells transfected with mutant
p53 give higher levels of p53 cross-reaction (data not shown).
Effects of Mutant p53 on G1 Arrest and Radiosensitivity after
Transfectioninto a RadiosensitiveCell Line ExpressingWild
Type p53. A p53 complementary DNA containing a mutation at codon
143, leading to a substitution of alanine for valine, expressed from a
CMV promoter, (14) was transfected into the radiosensitive human
3719
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CELL CYCLE ARREST AND RADIOSENSITIVITY
200 -
A. r—O.SO$,
p'cO.Ol
200 -
B. r—O.795,
p<O.O1
U
150-
150-
I
.5
U
.5
@
U
100III
U
@
m@_
U'
U
z
U
U'
Lu
U
Lu
a.
Lu
a.
50 -
50
0
0
0
@
@
0@
Fig. 1. Radiation-induced cell cycle arrest and
radiosensitivity. A, the surviving fraction at 2 Gy
(SF2Gy) plotted against the percentage of BrdUrd
positive cells 24 h after 2 Gy irradiation. B, SF2Gy
0
0
—
I
0@
0.0
plottedagainstthe percentageof BrdUrd-positive
0.2
0.4
0.6
0.8
0.0
cells 24 h after irradiation with a dose giving 0.2—
SF2Oy
C. r—O.29,p-NS.
0.3 surviving clonogenic fraction. C, SF2Gy plot
tedagalnstthemaximalpercentageofcellswith4N
DNA after 2 Gy irradiation.D, SF2Gyplotted
againstthe maximalpercentageof cells with 4N
500 -
I
1.0
I
0.2
0.4
0.6
0.8
1.0
SF2Gy
500 -
D. r-O.414,
p-N8.
0
U
DNA 24 h after irradiation with a dose giving
0.2—0.3
survivingclonogenicfraction.0, radiosen
sitive lines; U, radioresistant lines. The Pearson's
400-
correlation coefficients (r) and the respective P
400-
valuesare shown.NS,notsignificant.
U
@
300-
U
@
0
0
0
5300-
U
‘U
U
0
Lu
U
@
Lu
U
0
200-
U
U
a.
U
U
@200-
0
U
Do
100-
@
00.0
100-
.
0.2
‘
0.4
.
,
0.6
0.8
SF2Oy
ovarian cell line A2780. The A2780 cells express only wild-typep53 (9),
and irradiation of the parental A2780 cell line and vector-alone transfec
tants showed that these lines possessed a G1 arrest and inhibited DNA
synthesis after irradiation (Fig. 3A). Two of the A2780 mutant p5.3
transfectants lost the G1 arrest and continued to incorporate BrdUrd after
irradiation. These two mutant p5.3 transfectantS expressed increased 1ev
els of p53 as detected by MAb24O EUSA, whereas one mutant p5.3
transfectant, which still possessed a G@arrest, did not show increased p53
expression by EUSA compared to vector-alone controls (data not
shown). This demonstrates that, in this ovarian cell line, expression of a
mutant p53 can abrogate G1 arrest in a dominant-negative manner,
confirming that wild-type p53 ftmction is necessary for the G@arrest in
these cells.
Radiation sensitivities of the mutant p53 transfectants, which have
been shown to lose the G@ arrest, and vector-alone controls were
compared by clonogenic assays after irradiation with an acute dose of
-y-rays. As shown in Fig. 3B, the mutant p53 transfectants of A2780
are more resistant to ionizing radiation than vector-alone transfectants.
0
1.0
0.0
I
I
0.2
0.4
0.6
0.8
1.0
SF2Gy
Using a linear quadratic model to fit the surviving fraction after
irradiation, the vector-alone transfectants have a parameters of 0.18
and 0.47 with SEs of 0.12 and 0.18, respectively, and fi parameters of
0.26 and 0.23 with SEs of 0.05 and 0.07, respectively. The mutant p53
transfectants have a parameters of 0.19 and 0.05 with SEs of 0.07 and
0.08, respectively, and @3
parameters ofO.09 and 0.12 with SEs of 0.02
and 0.03, respectively. Thus, the mutant p53 transfectants of A2780
cells were significantly more resistant to an acute exposure to ionizing
radiation in the dose range 1—3(ly than vector-alone controls or the
parental A2780 cells, and the difference in sensitivity is particularly
apparent by a reduction in the f3 parameter of the fit to a linear
quadratic model. Therefore, abrogation of a radiation-induced G1
arrest in these radiosensitive cells decreases their radiosensitivity.
Discussion
We have shown a correlation between intrinsic radiosensitivity of
human tumor cell lines and the level of G1 arrest induced by ionizing
3720
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CELL CYCLE ARREST AND RADIOSENSITIVITY
Radiosensitive
lines:
SKNSH
F-
—
@
+
HX142
1F@
—
NB1
11
+
—
SuSa
+
II
Fig. 2. p53 levels after irradiation. The levels of
—
GCT27
‘F
+-.
• •
A2780
ir
+
—
•
P53
p53 were detected by immunoblotting in the cell
lines 8 h after treatment with 2 Gy ‘y-rays
(+) or
after no treatment (—).
‘.
Radioresistant
lines:
CCM
@
@
+
F__
U251
iF
UVW
1l_
radiation. Cells possessing a G1 arrest are more sensitive to ionizing
radiation than cells lacking such an arrest. Similar correlations have
been made in Burkitt's lymphoma cell lines and in ovarian lines
selected for resistance in vitro to DNA-damaging agents (8, 9). We
observed no correlation with the level of G2 arrest in the lines,
although this does not exclude the possibility that, for any given line,
G2 arrest may affect sensitivity. We have used cell lines that have a
wide range of radiosensitivity (SF2Gy, 0.1 1 to 0.8) and which include
cell lines derived from tumors which are particularly radiosensitive.
This may explain why correlation between radiosensitivity and p53
mutation has not been observed in previous studies in other tumor
lines with narrower sensitivity ranges (19), although it is also now
clear that p53 function can be disrupted by factors other than muta
tions in the p53 gene (15). Indeed, measuring G1 arrest induced by
DNA-damaging agents such as ionizing radiation may be one of the
most effective means of assaying p53 function.
It has previously been shown that mammalian radiosensitive mutant
cell lines often lack G1 arrest (3). Furthermore, increased accumula
tion of p53 protein after DNA damage can be defective in certain cell
lines derived from individuals who are sensitive to DNA-damaging
agents (1, 7). It has been suggested that radiosensitive tumors may
possess similar types of genetic changes to these radiosensitive mu
tants (1, 7). However, we observed no evidence of this in the radio
sensitive tumor lines examined, as all possess a G1 arrest and all
increase levels of p53 after irradiation.
Expression of mutant p53 (codon 143, Val to Ala) in the radiosen
sitive cell line A2780, which normally only expresses wild-type p53,
causes the cells to lose the radiation-induced G1 arrest. These mutant
p53 transfectants are more resistant than the parental or vector-alone
transfectants to an acute exposure of ionizing radiation as measured
by clonogenic assay. These results do not show that absence of a G1
arrest confers resistance, rather they show a correlation between p53
function (as measured by G1 arrest) and radiosensitivity. The loss of
p53 function may have other effects on the cells, including loss or
reduction in the kinetics of apoptosis induced by ionizing radiation.
This possibility would be in agreement with the observations that
genetic inactivation of the p5.3 gene in transgenic mice causes in
creased resistance of normal cells to ionizing radiation-induced apop
tosis (10). However, abrogation of a G1 arrest in colon tumor cells by
expression of a dominant negative mutant ofp53 (1 1) has been shown
previously to have no effect on radiosensitivity. This may be due to
colon cells having other resistance mechanisms operating that over
whelm any effect of loss of p53 function. Such mechanisms may
include increased bcl-2 expression and expression of other genes
which modulate apoptosis (20). This implies that the impact of p53
function on radiosensitivity will vary between different tumor types,
depending on the expression of other components of a DNA damage
inducible apoptotic pathway.
T98G
1 F_
Ui
SBI8
1 I@_@
ISO -
IF
RT112
1F__
1
A.
-
R
-
-
‘U
I @100.
U-
-
a. 50 -
0-0
I
10
I
20
TIME
I
30
40
50
(HOUMS)
1.0
0.0
•1.0
.E -2.0
-3.0
-4.0
0.0
1.0
2.0
3.0
DOSE (Gy)
4.0
Fig. 3. Radiation-induced G1 arrest and radiosensitivities of A2780 p53 transfectants A,
the percentageof BrdUrd-positivecells after irradiationwith 2 Gy. Points means of at least
two independent experiments counting a minimum of 20,000 events@B, the surviving fraction
ofclonogenic cells,expressedas naturallog,after irradiation.Linesare fittedusing a two-order
polynomial regression; bars SD. U vector-alone transfectants ofA2780 cells; U, mutantp53
(codon 143, Val to Ala) transfectants; @,
A2780 parental cells.
3721
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CELL CYCLE ARREST AND RADIOSENSITIVITY
The fact that radiosensitive cell lines possess functional p53 paral
lels observations in human tumors. The majority of neuroblastoma
and testis tumors are highly responsive to both radiotherapy and
chemotherapy and have been shown to express wild-typep53 (21, 22).
This would predict that tumors that have mutant p53 will be radiore
sistant; however, because p53 function can be abrogated by a number
of mechanisms, the presence of wild-type p53 will not necessarily
predict sensitivity. Instead, it will be necessary to fmd suitable means
of examining p53 function in tumor biopsies. Utilization of the pres
ence of a functional G1 arrest in cells during radiotherapy or chemo
therapy may be one such means.
ing UV or ionizing radiation: defects in chromosome instability syndromes? Cell, 75:
765—778,1993.
8. O'Connor, P. M., Jackman, J., Jondle, D., Bhatia, K., Magrath, I., and Kohn, K. W.
Role of the p53 tumour suppressor gene in cell cycle arrest and radiosensitivity of
Burkitt's lymphoma cell lines. Cancer Res., 53: 4776—4780, 1993.
9. Brown,R.,Clugston,C.,Burns,P.,Edlin,A.,Vasey,P.,Vojtesek,B.,andKaye,S. B.
Increased accumulation of p53 in cisplatin-resistant ovarian cell lines. Int. J. Cancer,
55: 1—7,
1993.
10. Clarke, A. R., Purdie, C. A., Harrison, D. J., Morris, R. G., Bird, C. C., Hooper, M. L,
and Wyllie, A. H. Thymocyte apoptosis induced by p53-dependent and independent
pathways. Nature (Lond.), 362: 849—852, 1993.
11. Slichenmyer, W. J., Nelson, W. G., Slebos, R. J., and Kastan, M. B. Loss of a
12.
Acknowledgments
13.
We thank Prof. B. Vogelstein (Baltimore, MD) for the plasmid pCS3-SCX3
and Prof. D. P. Lane (Dundee, United Kingdom) for p53 antibodies. We also
would like to thank S. Oakes and Prof. T. Cooke (Glasgow, United Kingdom)
for advice with flow cytometry and Dr. T. Wheldon (Glasgow, United King
dom) for statistical analysis of survival curves. We are grateful to Prof. S. B.
Kaye,
Glasgow,
manuscript
United
Kingdom,
for advice
and S. Jenkins for technical
during the preparation
14.
15.
of the
16.
assistance.
Med.Chir.,32: 725—732,
1993.
17. Davidoff,A. M., Pence,J. C., Shorter,N. A., Igiehart,J. D., and Marks,J. R.
References
1. Kastan, M. B., Zhan, 0., 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
checkpointpathwayutilizingp53andGADD4Sis defectivein ataxia-telangiectasia.
Cell, 71: 587—597,1992.
2. Weinert, T. A., and Hartwell, L. H. Characterisation of RAD9 of Saccharomyces
cerevisiae and evidence that its function acts post-translationally in cell-cycle arrest
Expression of p53 in human neuroblastoma- and neuroepithelioma-derived cell lines.
Oncogene, 7: 127—133,1992.
18. Wolf, D., and Rotter, V. Major deletions in the gene encoding the p53 tumour antigen
cause lack of p53 expression in HL-60 cells. Proc. NatI. Acad. Sci. USA, 82:
790—794,
1985.
19. Brachman, D. G., Beckett, M., Graves, D., Haraf, D., Vokes, E., and Weichselbaum,
R.R.p53mutationdoesnotcorrelatewithradiosensitivity
in24headandneckcancer
after DNA damage. Mol. Cell. Biol., 10: 6554—6564,1990.
3. Thacker,J., andGanesh,A. N. DNAbreakrepair,radioresistance
of DNAsynthesis
and camptothecin sensitivity in the radiosensitive irs mutants: Comparison to ataxia
telangiectasia cells. Mutation Res., 235: 49—58,1990.
4. Fertil, B., and Malaise, E. P. Inherent radiosensitivity as a basic concept for human
tumour radiotherapy. Int. J. Radiat. Oncol. Biol. Phys., 7: 621—629,1981.
cell lines. Cancer Res., 53: 3667—3669, 1993.
20. Sentman, C. L., Shutter, J. R., Hockenbery, D., Kanagawa, 0., and Korsmeyer, S. J.
bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes.
Cell, 67: 879—888,1991.
21. Komuro, H., Hayashi, Y., Kawamura, M., Hayashi, K., Kaneko, Y., Kamoshita, S.,
5. Parris, C. N., Mett, C. F., Lehmann, A. R., Green, M. H., and Masters, J. R.
Differential sensitivities to y radiation of human bladder and testicular tumour cell
lines. Int. J. Radiat. Biol., 53: 599—608, 1988.
6. Steel, G. G., McMillan, T. J., and Peacock, J. H. The picture has changed in the 198th.
Int. J. Radiat. Biol., 56: 525—537,1989.
7. Lu, X., and Lane, D. P. Differential induction of transcriptionally
p53-associated G1 checkpoint does not decrease cell survival following DNA dam
age. Cancer Res., 53: 4164—4168, 1993.
Vojtesek, B., Bartek, J., Midgley, C. A., and Lane, D. P. An immunochemical
analysis of the human nuclear phosphoprotein p53: new monoclonal antibodies and
epitope mapping using recombinant p53. J. Immunol. Methods, 151: 237—244,1992.
Gannon, J. V., Greaves, R., Iggo, R., and Lane, D. P. Activating mutations in p53
produce a common conformational effect: a monoclonal antibody specific for the
mutant form. EMBO J., 9: 1595—1602,1990.
Baker, S. J., Markowitz, S., Fearon, E. R., Wilson, J. K. V., and Vogelstein, B.
Suppression of human colorectal carcinoma cell growth by wild type p53. Science
(Washington DC), 249: 912—915,1990.
Vogelstein, B., and Kinzler, K. W. p53 function and dysfunction. Cell, 70: 523—526,
1992.
Tabuchi, K., Fukuyama, K., Mineta, T., Oh-Uchida, M., and Hori, K. Altered
structure and expression of the p53 gene in human neuroepithelial tumours. Neurol.
Hanada, R., Yamamoto, K, Hongo, T., Yamada, M., and Tsuchida, Y. Mutations of
the p53 gene are involved in Ewing's sarcomas but not in neuroblastomas. Cancer
Res., 53: 5284—5288,1993.
22. Peng, H., Hogg, D., Malkin, D., Bailey, D., Gallie, B. L., Bulbul, M., Jewett, M.,
Buchanan, J., and Goss, P. E. Mutations of the p53 gene do not occur in testis cancer.
active p53 follow
Cancer Res., 53: 3574—3578, 1993.
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Cell Cycle Arrests and Radiosensitivity of Human Tumor Cell
Lines: Dependence on Wild-Type p53 for Radiosensitivity
Amanda J. McIlwrath, Paul A. Vasey, Gillian M. Ross, et al.
Cancer Res 1994;54:3718-3722.
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