[CANCER RESEARCH 54, 3939—3946, July 15, 19941
Lack of a Correlation between Radiosensitivity and DNA Double-Strand
Induction or Rejoining in Six Human Tumor Cell Lines'
Break
Peggy L Olive,2Judit P. Banäth,and H. Susan MacPhail
British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada V5Z 1L3
ABSTRACT
to remove proteins, when subjected to an electric field. This method
is similar to conventional pulsed-field gel electrophoresis methods
The neutral filter elution method and the neutral comet assay have been which measure the amount of DNA remaining in the plug after
used to analyze radiation-Induced
DNA damage and repair in 6 human
electrophoresis, but this method has the important advantage of mea
tumor cell lines: HT-144 melanoma; DU-145 prostate carcinoma; U-87
suring DNA damage in individual cells. Gel electrophoresis methods
glioma; WiDr and HT-29 colon adenocarcinomas;
and SIHa cervical
are as sensitive as and less technically demanding than neutral-filter
carcinoma. In spite of large differences in intrinsic radiosensitivity
mess
elution,
although results using both methods are complicated by
ured using a clonogenic assay, double-strand
break induction, rejoining
retardation,―the much slower migration and elution of DNA
rate, and amount of residual DNA damage 4 h after Irradintion were “S-phase
from S-phase cells (14, 32—34),apparently as a result of the presence
similar among these cell lines when measured using the neutral comet
assay. Differences in initial numbers of double-strand
breaks were ob
of replication forks which retard DNA migration or elution (35). The
served using the neutral filter citation method; however, there was no ability of the comet assay to measure not only DNA damage but also
correlation with radiosensitivity, nor did the rejoining rate or amount of DNA content of the same cell (19, 35) provides a simple way of
residual DNA damage measured using filter elutlon correlate with mdi
identifying cells in various phases of the cycle, thereby avoiding this
ation sensitivity.
We conclude
that neither double-strand
break assay is
problem. While none of the studies summarized in Table 1 report
able to reliably rank cells according to clonogenic survival following
differences in DSB induction using neutral gel electrophoresis, it
irradiatIon.
would appear that some radiosensitive cell types can be identified on
the basis of a slower DSB rejoining rate.
INTRODUCTION
Because of the inconsistencies apparent in Table 1, we have exam
med
DNA damage using both the neutral filter elution and the neutral
Initial numbers of DNA DSBs3 induced by ionizing radiation
comet
assays using 6 established human tumor cell lines. DNA
and/or rates of rejoining of DSBs have been shown to correlate in
some studies with tumor cell radiosensitivity (Table 1, Refs. 1—28). induction, rejoining, and residual damage were measured and com
pared with the accepted standard of cell radiosensitivity and clono
These results suggest that it may be possible to predict the intrinsic
genic potential.
radiosensitivity of cells from individual human tumors using one or
more assays that detect DSBs. Knowledge of intrinsic radiosensitivity
of cells from tumor biopsies, if obtained in a timely fashion, could be
MATERIALS AND METhODS
used to optimize radiotherapy protocols with the goal of improving
Cell CultUresand IrradiationProcedure.All tumorcell lines were
individual tumor response to treatment.
obtained
from American Type Culture Collection, Rockville, MD. Cells were
While (unrepaired) DSBs are believed to be critical to radiation
adapted and maintained for 4—6months prior to experimentation as exponen
induced cell death, the ability to use either numbers of DSBs induced
tially growing monolayers in minimal essential medium with Earle's salts
by a given dose of radiation or rate/extent of rejoining to predict
(GIBCO Canada, Burlington, Ontario, Canada) supplemented with 10% fetal
intrinsic radiosensitivity remains controversial. For the neutral filter
bovine serum and antibiotics
(Sigma Chemical Co., St. Louis, MO). For cell
elution method, radiation sensitivity has been shown to correlate with
survival experiments, exponentially growing cells were removed from dishes
initial DNA damage, with DSB rejoining rate, with both, or with
using 0.1% trypsin from GIBCO and resuspended at a density of 5 X 10@
neither (Table 1). There appears to be a reasonable consensus, how
cells/mI. Cells were irradiated on ice and then plated at a density appropriate
to give about 300 surviving cells/9-cm tissue culture dish. Lethally irradiated
ever, that neutral ifiter elution detects, in addition to DSBs, other
properties
of DNA
organization
intrinsic
to the cell. Differences
in
DNA “packaging―
appear to influence the amount of DNA able to
elute through the filter in a given period of time, and these same
differences
could
be
relevant
to the
efficiency
of
DNA
cells ofthe same celltype (7 X 10@'/9cm-dish)were
as feeders, and colonies were stained approximately
Survival data were fitted to the linear-quadratic equation
repair
ln S=
(2, 29—31).
To supplement the neutral filter elution method, we have developed
the neutral comet assay to detect DSBs (19). The comet assay mea
sures the mobility of DNA from cells, embedded in agarose and lysed
added to each dish to serve
2 weeks later and counted.
J@f3J@2
where S is the surviving fraction and D is the radiation dose.
Irradiation was performed with cells at 4°Cusing an X-ray machine oper
ated at 250 kV at a dose rate of 3.2 Gy/min. Rejoining studies were performed
by irradiating
cells on ice and then diluting
cells in medium
at 37°C for the
specified repair time. For repair times longer than 10 mm, cultures were
Received 3/25/94; accepted 5/17/94.
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.
1 This
work
was
supported
by
a grant
from
the
National
Cancer
Institute
of
whom
requests
for
reprints
should
be
addressed,
at
the
Medical
Biophysics
Department, British Columbia Cancer Research Centre, 601 W. 10th Ave., Vancouver,
British Columbia, Canada, V5Z 1L3.
3 The
abbreviations
used
are:
DSB,
double-strand
break;
SDS,
sodium
dodecyl
sulfate;
a and f3, constants of the linear and quadratic terms in the linear-quadratic equation
S =
describing survival (5) as a function of radiation dose (D).
time.
Neutral
Comet
Assay.
Single
cells were centrifuged
and resuspended
in
ice-cold phosphate-buffered saline at a concentration of 3 X iO@cells/mi.
Following irradiation or drug treatment, 0.5 ml of cell suspension was placed
Canada
with funds provided by the Canadian Cancer Society.
2 To
returned to tissue culture dishes and trypsinized for 5 mm at the stated repair
in a 5-mi disposable
(Sigma
tube, and 1.5 ml of 1% low-gelling
type VII dissolved
in distilled
water
temperature
agarose
and held at 40°C) were added
to
the tube. The contents were mixed and 1.5 ml were pipetted quickly onto a
fully frosted microscope slide and allowed to gel for about 30 s on a cold
surface. Slides were carefully submersed
in lysing solution consisting
of 30
mMEDTA and 0.5% SDS (pH 8.3), and the temperature was raised to 50°C
for 4 h by placing containers with slides into an oven. Slides were then
3939
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RADIOSENSITIVITY
AND DNA DOUBLE-STRAND
BREAKS
electrophoresisCorrelation
Table 1 Comparisons between ceII lines varying in radiosensitivity using nesaral filter elution and neutrci gel
between
radiosensitivity and
double-strand breakCell
linesRef.Induction
RejoiningNeutral
elutionYes
filter
No
(28)a
not
Yes
L5178Y-R and -S mouse lymphoblast
Wlodek and Hittelman (6)
Yes
Yes
Yes
Yes
Yes
6 human squamous cell carcinoma
5 human cervix carcinoma cell lines
2 human neuroblastoma cell lines (HX142, 138)
L5178Y-S and -R
Schwartz et al. (7)
Kellend et aL (8)
McMillan ci aL (9)
Evans et aL (10)
CHO, xrs-1, xrs-5
5 mammalian cell lines
Dahm-Daphi et aL (11)
Eguchi-Kasai et aL (12)
Yes
Yes
Yes
Yes
No
No
No
Yes
No
No
Yes
ND
No
No
No
No
ND
ND
No
No2
6 human B-lymphoblast cell lines
Evans et aL (13)
Mouse mammary 66, 67 cell lines
9 human tumor cell lines
Sweigert et al. (14)
McMillan et aL (15)
CHO and xrs-5
Iliakiset aL (16)
3 CHO cell lines (AA8, EM9, NM2)
(18)Neutral
3 hamster
cell lines (V79, irs-i, irs-2)Oshita
Jones et aL
et aL (19)
No
No
V79, CHO, L5178Y-R
2 squamous cell carcinomas
No
No
H-ras/V-myc
No
No
V79, irs-i, irs-2, irs-3
No
No
No
No
Yes
Yes
V79 and Caski human squamous cell carcinoma
L5178Y-S and -R
No
No
No
No
Yes
Yes
Yes
YesHamster
CHO,
Chinese
hamster
fliakis et al. (21)
Cheong et aL (22)
Flentje et aL (23)
Olive et aL (19)
Giaccia et aL (24)
Kysela et aL (25)
Waiters et al. (26)
Iliakis et aL (27)
5 human squamous carcinoma cell lines
V79 cells and a sensitive subline
Mouse L-cells and 2 sublines
CHO and xrs-5
Human colorectal cells: HT29, SW48Olive
Lambin et aL
ovary.
overnight
followed by electrophoresis
mM boric
acid
at 0.55
Smeets et aL (20)
transformed rat embryo cells
washed free of detergent by transferring them to a container with a large
volume of 90 mM Tris-2 mM EDTA-90 mM boric acid buffer (pH 8.5)
buffer
Van Ankeren et aL (17)
gel
No
done;
Radfordand Hodgson(2)
Wiodek and Olive (3)
Schwartz et aL (4)
Green et aL (5)
ND°
ND
ND
ND
electrophoresisNo
@
et aL (1)
human lung cancer cell lines
Hamster V79, mouse L-cells
V79, CHO, mouse L5178Y-R, L5178Y-S
8 human squamous cell carcinoma cell lines
2 CHO cell lines (EM9, AA8)
Yes
Yes
Yes
Yes
V/cm
in 90 m@iTris, 2 m@tEDTA, and 90
for 25 mm.
Slides
were
rinsed
and
(pH 9.6). Elution was performed for 10 h with 2 mMEDTA, 20 simsTris, and
0.2% SDS using a flow rate of 0.04 mi/mm. The radioactivity of the samples
was measured in an LKB 1219 liquid scintillation counter after adding 6 ml of
Hydrofluor scintillation fluid (National Diagnostics, Manville, NJ). Results are
stained for 1 h in 2.5 @.tg/ml
propidium iodide. To prevent detachment of the presented as absolute radioactivity remaining on the filter or as radioactivity
agarose after lysis, slides were often placed for about 2 mm in the 50°C relative to an internal standard.
oven and the edges were allowed to dry. Individual cells or “comets―
were
viewed using a Zeiss epifluorescence microscope and image analysis
system (36). Double-strand damage was quantified as an increase in tail RESULTS
moment, the product of the amount of DNA (fluorescence) in the tail and
the distance between the means of the head and tail fluorescence distribu
Cell Survival. Radiationsurvival curves for the six humantumor
lions. For each comet, tail moment and total DNA content (total fluores
cell lines are shown in Fig. 1; plating efficiency and survival param
cence per comet in arbitrary units) were recorded. In some experiments, the eters, a, ,3, and the surviving fraction after 2 Gy irradiation are given
response of G@and 02 cells only was determined on the basis of DNA
in Table 2. These cell lines encompass a wide range of radiosensitiv
fluorescence by using data from comets with relative DNA content values
ities with HT29 and its derivative, WiDr, representing the most
greater than 20 and less than 10.
radioresistant
and HT-144 representing the most radiosensitive cell
Nondenaturing
Filter Elutlon.
Nondenaturing
filter elution was a modi
fication
of the method
radiolabeled
of Bradley
with 0.74 kBq/ml
and Kohn
(37).
[‘4Cjthymidine
DNA
(specific
of test cells
activity,
lines.
was
NeutralCometAssay.Resultsusingthecometassayshowfairly
2.07 GBq/
mmol; Amersham) for two cell doublings (24—48h). Following radiolabeling,
fresh medium was added to the cultures for 4 h. Cells were irradiated and then
mixed with irradiated “internal
standard―
cells, Chinese hamster V79 cells that
had been radiolabeled for 24 h with 3.7 kBq/ml [3H]thymidine (specific
conclusively that there is no significant difference in the initial num
ber of DSBs detected for these cell lines (Fig. 2, i—n).Even when
analysis
was confined
to non-S-phase
cells,
there
was
no relation
Reproducibility between filters was excellent, so that results could be pre
sented as absolute counts per fraction as well as elution relative to the internal
between the initial numbers of breaks and radiosensitivity (Fig. 2/i).
DNA content varied considerably between cell lines but apparently
had no influence on amount of damage induced or detected. Bivariate
plots of DNA content versus tail moment for DU-145 and HT-144
cells show the dramatic influence of active replication forks on DNA
standard.
migration
activity,
925 GBq/mmol;
Amersham).
Preliminary
studies
indicated
a coelu
tion problem between the test and internal standard cells which could be
minimized by limiting the total cell number to less than 5 X 10@cells/filter.
The cell mixture (5 x 10@cells total) was layered onto 25 mm (2 @m
pore
size) Nucleopore polycarbonate
filters, washed twice with ice-cold phosphate
buffered saline, and lysed with 5 ml of 2 mMEDTA, 1% SDS, and 20 mMTris
from cells with S-phase
DNA content(Fig.
3). However,
for
a given dose of radiation, there was no apparent difference in the
pattern or amount of damage produced in these two cell lines or in any
of the other tumor cell lines examined.
3940
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RADIOSENSITWITY
AND DNA DOUBLE-STRAND
@
I
BREAKS
‘
,
I
‘
‘
‘
,
I
1 @°
0
.‘@
1I@-1
C)
CD
UD)
@
Fig. 1. Clonogenicity of six human tumor cell
-2
•
lines following exposure to X-rays. Symbols,
means ±SD (bars) for 4—6independent experi-
1 0
•U87
.
ments.
>
C/) io-3
V
HT29
V
WiDr
0
Du145
U
HT144
L@
SiHa
io@
0
5
10
Radiation
Rejoining of DSBs was also independent of cell type, as shown in
Fig. 2. Less than 10% of the initial number of breaks remained 4 h
after 50 Gy, and this value was not significantly different for the six
tumor
cell lines.
Of the rejoinable
breaks,
approximately
15% were
therefore accumulate more rapidly in G2 following irradiation. The
dotted lines for G1 cells only in Fig. 2, a—findicate the effect is small
and may become important only when assessing residual DNA dam
age 4 h after treatment.
still present 1 h following 50 Gy. The time to rejoin one-half of the
breaks averaged about 14 mm (Table 2) and did not differ significantly among the cell lines examined. Bivariate plots indicated that
Dose (Gy)
Results
shown
in Fig. 2g and the values
for
residual damage in Table 2 therefore represent the response of the G1
and G2 cells only.
Neutral Filter Elution. Two methods were used for quantifying
DSB rejoining rates were independentof cell cycle position for the
the amountof DNA eluted. The absoluteamountof DNA eluted from
tumor cell lines (Fig. 4). However, radiation-induced increases in the
duration of S phase (inhibition of replicon initiation and chain dongation) can lead to an apparent increase in the rate of rejoining. This
is more obvious in the data for V79 cells which cycle faster and
the filter 10 h after irradiation was not significantly different among
the human tumor cell lines tested, with the possible exception of the
SiHa cells which appeared to elute more slowly (Fig. 5a). However,
averaging results between experiments tends to mask small differ
Table 2 Radiation survival cutee parameters, DSB induction rates,
rejoiningkineticsDamage
and DSB
inductionRepair
half-timeResidualdamageCell
@eCome1NFE1CometDU145
lineaSF21'PECComet―
prostatic carcinoma
SiHacervicalcarcinoma—0.06
(0.04)1ff144
(0.06)
(0.013)
—0.31
—0.025
(0.14)—0.055 (0.016)0.61
(0.08)
0.41
(0.07)0.50
(0.11)
0.59
(0.06)0.15
(0.13—0.16)
0.15
(0.12—0.17)
(0.15)—0.010 (0.020)0.09
(0.02)0.38
(0.07)0.16
(0.13—0.16)
(0.04)—0.043(0.008)0.76
(0.08)0.70
(0.06)0.15 (0.12—0.16)
(0.014)0.84
(0.04)0.61
(0.09)0.14 (0.12—0.15)
(2.8)
13.0
(3.1)9.2
(4.1)
12.6
(3.6)0.06
(0.03)
0.04
(0.05)0.07
(0.12)13.2
1.41
(0.08)14.5
1.35
(0.06)13.1
1.22
(0.05)
(4.4)6.9
(0.4)0.05
(0.02)0.03
(1.6)7.0
(2.5)0.05
(0.04)0.07
(2.6)6.9
(0.6)0.08
(0.02)0.08
(4.4)
(0.5)
1.0015.7
13.36.7
(0.06)
0.030.03
(0.05)
0.14
1.23
melanoma—1.20
(0.01)WiDr
colon carcinoma—0.04
(0.03)HT29
colon carcinoma—0.04
(0.02)0.045
(0.05)U87
glioma
(0.03)
(0.11)
(0.008)
0.07(Chinese
—0.16—0.023
—0.0180.43 0.680.17
V79lungfibroblast—0.37
hamster)(0.08)(0.009)(0.06)(0.06)(0.15—0.18)(2.5)(1.8)(0.02)(0.03)
@
1.12
(0.09)
1.09
(0.09)9.8
(0.07)
(0.11—0.15)
0.16
0.790.13
8.00.10
(0.02)
a and valuesdetermined
fromthelinear-quadratic
equation
describing
clonogemc
survival
asafunction
ofradiation
dose.Mean(SD)for4—6
survival
curves.
5@ @sthe fraction of cells surviving exposure to 2 Gy. Mean (SD) for 6—10experiments.
CPE,
plating
efficiency
mean
(SD)
for8—10
experiments.
d Slope
a Relative
of dose-response
amount
of DNA
curve
eluted
(95%
from
confidence
filters
limits)
by
from
10 h after
Fig.
exposure
2k
of cells
to 75
Gy.
Mean
(SD)
for
12 filters.
Elution
is relative
to the
internal
standard
V79
cells
which
also
received75 Gy.NFE,neutralfilterelution.
“Time
(mm) required to rejoin one-half of the initial number of double-strand breaks after exposure to 50 Gy for the comet assay or 25 Gy for NFE. Values represent the mean
(SD) of at least 3 independent experiments for rejoining rates calculated for the first 15 min of repair. Rejoining data are shown in Figs. 2 and 5.
g Residual damage is the fraction of initial damage remaining in cells 4 h after exposure to 50 Gy for the comet assay or 4 h after 25 Gy for NFE. The mean (SD) of 3 independent
experiments is shown. For the comet data, S-phase cells were excluded from analysis.
3941
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RADIOSENSITIVITYAND DNA DOUBLE-STRAND BREAKS
Q)
E
0
F-0
0
2
40
2
40
2
40
2
40
2
40
2
4
Time After Irradiation (h)
C)
C
h.
15
C
C
a,
E
E
a,
10
0
a,
C)
a,
a,
I—
5
a,
0
0
1
2
3
4
.
I
.
,
0
Time After Irradiation (h)
@
.
.
I
.
25
.
.
.
I
.
.
.
.
50
I.
75
Dose (Gy)
10
I—
0 25 50 75
0 25 50 75
0 25 50 75
Radiation
0 25 50 75
0 25 50 75
0 25 50 75
Dose (Gy)
Fig. 2. DSB induction and rejoining after 50 Gy for 6 human tumor cell lines measured using the comet assay. Average tail moment, proportional to the number of DSBs per cell,
was calculated from 100—200comets/point using a fluorescence image processing system. a—fresponse of asynchronous cells [symbols, mean ±SD (bars) for 3 experiments,
response of G1 and G2 cells onlyj. g compares the normalized rejoining response of G, and G2 cells from the different cell lines. h, DSB induction for G1 and 02 cells only. i—n,
means ±SD (bars) for asynchronous cells from 3 independent experiments.
ences in elution. Therefore, to improve sensitivity, each cell line was
first compared to an internal standard of Chinese hamster V79 cells
irradiated with the same dose as the test cells. To verify accurate
detection of the 3H- and ‘4C-labeledDNA, cross-over experiments
were performed where each cell line was labeled with [‘4C]thy
midine and [3H]thymidine. Samples were then mixed (3H-labeled
test cells mixed with ‘4C-labeled internal standard cells and vice
versa), eluted, and counted. As shown in Fig. 6, WiDr cells eluted
significantly more rapidly than the V79 cells and slightly more
rapidly than the U87 cells, but SiHa cells showed a similar rate of
elution as V79 cells. Based on similar analyses, Table 1 compares
the ratio of amount of DNA eluted in 10 h for the test cells relative
to the V79 cells. While the internal standard method increases
sensitivity, there was no apparent correlation between initial dam
age and surviving fraction for these 6 cell lines (Table 1).
Rejoining rates measured using neutral filter elution were com
parable among the cell lines with the exception of the SiHa cells
which showed less rapid rejoining and more residual damage than
the other cell lines (Fig. 5b). On average, about 7% of the initial
damage remained 4 h after 25 Gy and the fraction of unrejoined
breaks was not correlated with radiosensitivity (Table 2).
DISCUSSION
Several properties of a tumor contribute to its response to radio
therapy. One of the most important is the sensitivity of the tumor cell,
removed from its environment, to killing by ionizing radiation. The
conventional colony formation assay used to estimate intrinsic radi
osensitivity of human tumor cell lines is limited by several factors
including, the number of weeks required to obtain an estimate of
tumor cell radiosensitivity and the relatively small proportion of cells
that will survive and grow in vitro. These and other limitations of
clonogenic assays have stimulated an interest in developing new
methods which would provide a measure of intrinsic radiosensitivity
in a much shorter period of time so that results could be considered in
the planning of treatment (38). Of course, any assay suggested to fill
this role must provide results which correlate well with relevant
clonogenic end points such as the surviving fraction after 2 Gy
irradiation.
Our earlier work led us to believe that the neutral filter elution
assay, but not the comet assay, might provide an indication of intrinsic
differences in radiosensitivity. It is clear from a number of studies that
results obtained using neutral filter elution can be influenced by many
3942
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RADIOSENSITIVITY
@
0 Gy
BREAKS
25 Gy
50 Gy
75 Gy
HT
30
C
20
E
10
0
10 Gy
AND DNA DOUBLE-STRAND
0
30
‘‘‘‘I''''I'''
-‘1.
I
I.•@,@7@s7:%@
F.‘‘‘I''.,I_.,',I,,,J,',G.I.•
H.‘‘‘‘I''''l''''
DU-145
20
Co
10
@
,,,I@•!!.!,@,
0
0
10
20
30 0
10
20
30 0
10
20
30 0
10
20
30 0
10
20
30
DNA Content
Fig. 3. Bivariate display of tall moment for damage induction versus DNA content (total image fluorescence) measured immediately following irradiation on ice. Results for 100—200
comets/done are shown. Note the inhibition of migration in comets with S-phase DNA content.
factors, in addition to the number of strand breaks present in the cell
(39—42).Most of the studies in Table 1 were conducted at pH 9.6, and
perhaps the increased sensitivity for detecting DSBs at a slightly
alkaline pH may improve the ability to resolve small differences in
response between cell lines. One study that was not included in Table
1 showed a correlation between radiosensitivity and levels of DSBs in
high-dose rate (43). In these experiments, it is not clear whether the
results reflect initial damage or rejoining rates, but this approach may
provide an additional degree of sensitivity which could identify some
radioresistant cell types. Our data also indicate reproducible and
significant differences in DNA elution which cannot be explained on
the basis of numbers of initial DSBs produced in a cell. As observed
4 humantumor cell lines irradiatedat low-doserate but not at previously(6), thosedifferencesmaybe magnifiedat low radiation
0 mm
i 0 mm
30 mm
60 mm
240 mm
10
V-79
1
C
‘‘‘‘I''''I''''
I
‘‘‘‘I''''I''''
I
J.
E
HT-144
0
@
io@j@@
I1rG@@
I
@Hj@
_______
CO
N
10
_______
‘a1
‘
DU-145
1
0
10
20
3O@
10
20
30 0
10
20
30 0
10
20
30 0
10
20
30
DNA Content
Fig. 4. Bivariate display of tail moment versus DNA content for rejoining of strand breaks following exposure of cells to 50 Gy. Cells were irradiated on ice and then returned to
a 37°Cincubator for repair. Results for 100—200comets/time point are shown. Note the accumulation of cells in G2 by 4 h following irradiation.
3943
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RADIOSENS@V@@Y
AND DNA DOUBLE-STRAND
BREAKS
C
80
B. After 25 Gy
C
CD
E
a)
1
@0
a)
0)
CD
E
CD
0
@
Fig 5. DSB induction (A) and rejoining after 25
Gy (B) measured using the neutral elution method.
Symhols@ means ± SD (bars) for 4—6damage
0.1
inductionexperimentsand 3 repair experiments.
‘I
, response of Chinese hamster V79 cells.
0
C
0
C-)
CD
U-
0
@
0
20
40
60
.
I
.
.
0
80
of DNA which
may be related
“packaging―
and in some cases to radiosensitivity,
variations
among
these human
.
.
.
2
doses. However, elution differences do not correlate with radiosensi
tivity for these cell lines. Therefore, while there are reproducible
in the elutabiity
1
.1
4
Rejoining Time (h)
Dose (Gy)
differences
,
to DNA
we have not seen
tumor cell lines which would
allow us
to rank them accurately for radiosensitivity (clonogenicity).
Results using the comet assay confirmed our previous work with
this method indicating that the initial number of DSBs following
irradiation is independent of cell type (19). An advantage of the comet
assay over the neutral-filter elution method is that S-phase cells can be
omitted from analysis by using total comet fluorescence as a measure
of DNA content. Since replicating DNA migrates inefficiently, a cell
population with a higher proportion of S-phase cells appears to show
less overall DNA damage following irradiation. However, whether or
not S-phase cells were included in the analysis, all cell lines showed
identical dose-response relationships.
DNA rejoining kinetics from comet assays is in good agreement
with neutral-filter elution data considering the differences in the initial
dose delivered (see below); about one-half of the breaks were rejoined
within 15 mm following 50 Gy and only about 5% of the initial
damage was detectable 4 h after 50 Gy. DSB rejoining rates measured
using the comet assay should be more accurate than population-based
1.0
\3H4P@H-V79
4-J
0.5
U-
C-SiHa
Fig. 6. Neutral filter elution analysis of human
@
tumor cells using Chinese hamster V79 cells as an
internal control. Each panel shows results from a
single filter in which V79 and human tumor test
cells, radiolabeled with either [3Hjthymidine or
[‘4C]thymidine, were irradiated with 75 Gy, lysed,
and elutedfrom filtersfor 10 h. The amountof
tumor cell DNA eluted by 10 h, relative to the
0
0
amount of V79 cell DNA eluted at this time, is
C)
shown in Table 2.
CO
U-
i o
0.5
0.0
0
5
10
0
5
10
0
5
ElutionTime (h)
3944
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10
RADIOSENSITIVITYAND DNA DOUBLE-STRAND BREAKS
methods like neutral-ifiter elution since degrading cells are not ob
served and apoptotic cells can be easily omitted from analysis (44).
This may explain the observation that in spite of the higher radiation
dose used to induce significant damage for the comet assay, residual
damage for this method 4 h after irradiation was as low as or often
lower than for the neutral-filter elution assay. None of the cell lines
examined here demonstrated apoptosis during the 4 h of repair time
following irradiation.
Results in Fig. 4 indicate that all cells of the population rejoin
breaks at a similar rate and that there was no evidence of a slowly
repairing population of cells. Unlike single-strand break rejoining
(35), there was no indication that G1 cells rejoined DSBs breaks more
rapidly than cells in G2. We did notice, however, that measuring
residual damage 4 h after 50 Gy could be influenced by the increase
in the percentage of cells in S phase following irradiation; since
S-phase DNA migrates inefficiently, there was a reduction in the
amount of damage detected leading to a small (but correctable)
underestimate in the amount of residual DSBs.
Differences in absolute rejoining rates or residual damage between
the two assays are likely to be a result of differences in the initial
radiation dose (25 Gy for neutral-filter elution versus 50 Gy for the
comet assay). Iliakis et a!. (45), using a variety of DSB assays
including neutral filter elution, measured a half-time of rejoining of
15—20 mm following
exposure
of Chinese
hamster
ovary cells to 50
Gy, but only 9—12mm following 25 Gy, in good agreement with the
results in Table 2. The higher pH of the neutral filter elution method
(9.6 versus 8.3 for the comet assay) may also allow detection of more
DSBs (e.g., those formed from two single-strand breaks or alkali
labile sites which are displaced farther apart on the double-stranded
molecule). Since such lesions are likely to be rejoined more easily
than frank DSBs, the rejoining rate could appear faster for filter
elution than for the comet assay.
As concluded by Evans et aL (13), proficiency in DSB rejoining
appears to be necessary but not always sufficient for radiation resist
ance. Our results support this conclusion and indicate that DSB
induction and rejoining, measured using these two DSB assays, are
not adequate indicators of intrinsic tumor cell radiosensitivity. It
would seem that a subset of radiosensitive cells can be detected
because of the slow rejoining of DSBs (e.g., xrs.5). Another subset of
cells may be identifiable on the basis of rapid initial elution from
filters during neutral filter elution (e.g., mouse L5178Y-S). However
misrejoining is responsible undoubtedly for a substantial third subset
of radiosensitive cells, and these are unlikely to be detected with either
of these DSB assays. Therefore, it appears that some measure of
fidelity of repair will be necessary to reliably identify radioresistant
tumor cells.
radiosensitivity of L5178Y-S and L5178Y-R cells. Radiat. Res., ii2: 146—155,
1987.
7. Schwartz, J. L, Rotmensch, J., Giovanazzi, S. M., Cohen, M. B., and weichselbaum,
R. R. Faster repair of DNA double-strand breaks in radioresistant human tumor cells.
hit. J. Radial. Oncol. Biol. Phys., 15: 907—912,1988.
8. Kellend, L R., Edwards, S. M., and Steel, 0. 0. Induction and rejoining of DNA
double-strand breaks in human cervix carcinoma cell lines of differing radiosensitiv
ity. Radiat. Res., ii6: 526—538,1988.
9. McMillan, T. 3., Eady, J. J., Holmes, A., Peacock, J. H., and Steel, G. G. The
radiosensitivity of human neuroblastoma: a cellular and molecular study. Int. J.
Radiat.Biol.,56: 651—656,
1989.
10. Evans, H. H., Ricanti, M., and Horng, M. Deficiency
in DNA repair in mouse
lymphoma strain L5178Y-S. Proc. NatI. Acad. Sci. USA, 84: 7562—7566,1987.
11. Dahm-Daphi, I., Dikomey, E., Pyttlik, C., and Jeggo, P. A. Repairable and non
repairable DNA strand breaks induced by X-irradiation in CHO K! cells and the
radiosensitive mutants xrsi and xrs5. tnt. J. Radiat. Biol., 64; 19—26,1993.
12. Eguchi-Kasai, K., Kosaka, T., Sato, K, and Kaneko, I. Repairability of DNA
double-strand breaks and radiation sensitivity in five mammalian cell lines. Int. J.
Radiat. Biol., 59: 97—104,1991.
13. Evans, H. H., Ricanti, M., Horng, M., Jiang, 0., Mend, J., and Olive, P. DNA
double-strand break rejoining deficiency in TK6 and other human B-lymphoblast cell
lines. Radiat. Res., i34: 307—315,1993.
14. Sweigert, S. E., Rowley, R., Warters, R. L, and Dethlefsen, L A. Cell cycle effect
on the induction of DNA double-strand breaks by X-rays. Radiat. Res., 116: 228—
244, 1988.
15. McMillan, T. J., Casoni, A. M., Edwards, S., Holmes, A., and Peacock, J. H. The
relationship of DNA double-strand break induction to radiosensitivity in human
tumour cell lines. Int. J. Radiat. Biol., 58: 427—438,1990.
16. Iliakis, G., Okayasu, R., and Seaner, R. Radiosensitive xrs-5 and parental CHO cells
show an identical DNA neutral filter elution dose-response: implications for a
relationship between cell radiosensitivity and induction of DNA double-strand breaks.
Int. J. Radiat. Biol., 54: 55—62,1988.
17. Van Ankeren, S. C., Murray, D., and Meyn, R. E. lnduction and rejoining of
y-ray-induced DNA single- and double-strand breaks in Chinese hamster AA8 cells
and in two radiosensitive clones. RadiaL Res., ii6: 511—525,
1988.
18. Jones, N. J., Stewart, S. A., and Thompson, L H. Biochemical and genetic analysis
of Chinese hamster mutants irsi and irs2 and their comparison to cultured ataxia
telangiectasia cells. Mutagenesis, 5: 15-23, 1990.
19. Olive, P. L., Wiodek, D., and Banath, J. P. DNA double-strand breaks measured in
individual cells subjected to gel electrophoresis. Cancer Res., 51: 4671—4676, 1991.
20. Smeets, M. F., Mooren, E. H., and Begg, A. C. Radiation-induced DNA damage and
repair in radiosensitive and radioresistant human tumour cells measured by field
inversion gel electrophoresis. tnt. J. Radiat. Biol., 63: 703—713,1993.
21. Iliakis, U., Metzger, L, Muschel, R. J., and McKema, W. 0. Induction and repair of
DNA double-strand breaks in radiation-resistant cells obtained by transformation of
primary rat embryo cells with the oncogenes H-ms and v-myc. Cancer Rex., 50:
6575-6579,1990.
22. Cheong,
N., Wang,
Y., Jackson,
M., and Iliakis,
0. Radiation-sensitive
irs mutants
rejoin DNA double-strand breaks with efficiency similar to that of parental V79 cells
but show altered response to radiation-induced 02 delay. Mutat. Res., 274: 111—122,
19fl
23. Flentje, M., Asadpour, B., Latz, D., and Weber, K. J. Sensitivity of neutral filter
elution but not PFGE can be modified by non-dab chromatin damage. tnt. J. Radiat.
Biol., 63: 715—724,1993.
24. Giaccia, A. J., Schwartz, J., Shieh, J., and Brown,
inversion gel electrophoresis to predict tumor cell
24: 231—238,1992.
25. Kysela, B. P., Michael, B. D., and Arrand, J. E.
initial DNA damage and repair of double-strand
J. M. The use of asymmetric-field
radiosensitivity. Radiother. Oncol.,
Relative contributions of levels of
breaks to the ionizing radiation
sensitivephenotypeoftheChinesehamstercellmutant,XR-V15B.PartI. X-rays.Int.
J. Radiat. Biol., 63: 609—616, 1993.
26. Warters, R. L, Lyons, B. W., Chen, D. J., and Sato, K. DNA damage processing in
a radiation-sensitive mouse cell line. Mutat. Res., 293: 91—98,1993.
27. lliakis, G., Mehta, R., and Jackson, M. Level of DNA double-strand break rejoining
in Chinese hamster xrs-5 cells is dose-dependent: implications for the mechanism of
radiosensitivity. hit. I. Radiat. Biol., ói:315—321,1992.
28.
REFERENCES
Lambin,
P., Cedervall,
B., Chavaudra,
N., Sadie,
C., Joiner,
M. C., and Malaise,
E. P.
Initial and residual number of X-ray induced DNA double-strand breaks in radiosen
sitive and radioresistant human tumor cell lines. Radiother. OncoL, 25: S5l, 1992.
29. Wlodek, D., Olive, P. L Physical basis for detection of DNA double-strand breaks
1. Oshita, F., Fujiwara, Y., and Saijo, N. Radiation sensitivities in various anticancer
drug-resistant human lung cancer cell lines and mechanism of radiation cross
resistance in a cisplatin-resistant cell line. J. Cancer Res. Clin. Oncol., ii9: 28—34,
1992
2. Redford, I. R., and Hodgson, 0. S. lasI@induced DNA double-strand breaks: use in
calibration of the neutral filter clution technique and comparison with X-ray induced
breaks. Int. J. Radial. Biol., 48: 555-566, 1985.
3. Wlodek, D., and Olive, P. L Neutral ifiter elution detects differences in chromatin
organization which can influence cellular radiosensitivity. Radiat. Res., 132: 242247, 1992.
4. Schwartz, J. L, Mustafi, R., Bcckett, M. A., Czyzewski, E. A., Farhangi, E., Ordina,
D., Rotmensch, J., and Weichselbaum, R. R. Radiation-induced DNA double-strand
break frequencies in human squamous cell carcinoma cell lines of different radiation
using neutral filter elution. Radiat. Res., 124: 326—333,1990.
30. w@sers,
to -y-radiation.
Part 1: Intrinsicradiosensitivity.tat. J. Radiat.Biol, 61: 465—472,
R. w.
Detection
of ionizing
radiation-induced
DNA
Radiat. Res., i24: 309—316,1990.
31. Olive, P. L DNA organization affects cellular radiosensitivity and detection of initial
DNA strand breaks. Int. J. Radiat. Biol., 62: 389—396,1992.
32. Radford, I. F., and Broadhurst, S. Enhanced induction by X-irradiation of DNA
double-strand breakage in mitosis as compared with S-phase V79 cells. Int. J. Radiat.
Biol., 49: 909—914,1986.
33. iliakis, 0. E., Cicilioni, 0., and Metzger, L Measurement of DNA double-strand
breaks in CHO cells at various stages of the cell cycle using pulsed field gel
sensitivities. hit. J. Radiat. Biol., 59: 1341—1352,1991.
5. Green, A., Prager, A., Stoudt, P. M., and Murray, D. Relationships between DNA
damage and the survival of radiosensitive mutant Chinese hamster cell lines exposed
R. L., and Lyons,
double-strandbreaksby filter elutionis affectedby nuclearchromatinstructure.
electrophoresis: calibration by means of
@I
decay. Int. J. Radiat. Biol., 59: 343—357,
1991.
34. Okayasu,
R., Bloecher,
dose-response
of DNA
D., and Iliakis, 0.
neutral filter elution
Variation
through the cell cycle in the
in X-irradiated
synchronous
CHO cells.
Int.J. Radiat.Biol.,53: 729—747,
1988.
1992.
6. Wlodek, D., and Hittelman, W. N. The repair of double-strand breaks correlates with
35. Olive, P. L, and Banith, J. P. Induction and rejoining of radiation induced DNA
3945
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RADIOSENSITIVITY
AND DNA DOUBLE-STRAND
BREAKS
single-strandbreaks“tail
moment―
as a functionof positionin thecellcycle.Mutat.
Res., 294: 275—283,1993.
36. Olive, P. L., and Banath, J. P. Growth fraction measured using the comet assay. Cell
Prolif., 25: 447—457, 1992.
37.
Bradley,
M. 0., and Kohn, K. W. X-ray induced DNA double-strand
break production
and repair in mammalian cells as measured by neutral filter elution. Nucleic Acids
Res., 7: 793—804,1979.
38. Brock, W. A., Baker, F. L., and Tofilon, P. J. Tumor cell sensitivities to drugs and
radiation, In: J. D. Chapman, L. J. Peters, and H. R. withers (eds.), Prediction of
Tumor Treatment Response, pp. 139—155.Elmsford, NY: Pergamon Press, Inc.,
strand break analysis by filter elution. Int. J. Radiat. Biol., 54: 739—747,1988.
42. Prise, K. M., Davies, S., and Michael, B. C. Non-linear dose-effect curve for DNA
double-strand breaks by low LET radiation: the effect of eluting buffer composition
on the measurement of breaks by filter elution technique. Int. J. Radiat. Biol., 56:
943—950,
1989.
43. Cassoni, A. M., McMillan, T. J., Peacock, J. H., and Steel, G. 0. Differences in the
level of DNA double-strand breaks in human tumour cell lines following low
dose-rate irradiation. Eur. J. Cancer, 284: 1610—1614,1992.
44.
39. Hutchinson, F. On the measurement of DNA double-strand breaks by neutral elution
(Letter to the Editor). Radiat. Res., i20: 182—186,1989.
40. lliakis, 0. The role of DNA double-strand breaks in ionizing radiation-induced killing
of eukaryotic cells. Bioessays, i3: 641—648,1991.
41. Koval, T. M., and Kazmar, E. R. Eluting solution composition affects DNA double
Olive,
P. L., Fraser,
G., and Banáth, J. P. Radiation-induced
apoptosis
measured
in
TK6 human B lymphoblast cells using the comet assay. Radiat. Res., i36: 130—136,
1989.
1993.
45.
Iliakis, 0., Blocher,
D., Metzger,
L., and Pantelias,
0. Comparison
of DNA
double
strand break rejoining as measured by pulsed field gel electrophoresis, neutral sucrose
gradient centrifugation, and non-unwinding filter elution in irradiated plateau-phase
CHO cells. Int. J. Radial. Biol., 59: 927—939,1991.
3946
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Lack of a Correlation between Radiosensitivity and DNA
Double-Strand Break Induction or Rejoining in Six Human
Tumor Cell Lines
Peggy L. Olive, Judit P. Banáth and H. Susan MacPhail
Cancer Res 1994;54:3939-3946.
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