Mutagenesis vol. 20 no. 2 pp. 131--137, 2005
Advance Access publication 22 March 2005
doi:10.1093/mutage/gei018
Radiosensitivity of lymphoblastoid cell lines with a heterozygous
BRCA1 mutation is not detected by the comet assay and pulsed field gel
electrophoresis
Kristina Trenz, Petra Schütz and Günter Speit
Universit€atsklinikum Ulm, Abteilung Humangenetik, D-89070 Ulm,
Germany
Lymphoblastoid cell lines (LCL) with a heterozygous
mutation in the breast cancer susceptibility gene BRCA1
have been repeatedly used to elucidate the biological consequences of such a mutation with respect to radiation
sensitivity and DNA repair deficiency. Our previous results
indicated that LCL with a BRCA1 mutation do not generally show the same chromosomal mutagen sensitivity in the
micronucleus test as lymphocytes with the same BRCA1
mutation. To further study the radiosensitivity of LCL with
a BRCA1 mutation, we now performed comparative investigations with the alkaline (pH 13) and the neutral (pH 8.3)
comet assay and pulsed field gel electrophoresis (PFGE).
These tests are commonly used to determine the repair
capacity for DNA double strand breaks (DNA-DSB). Six
LCL (three established from women with a heterozygous
BRCA1 mutation and three from healthy controls) were
investigated. Induction (2 and 5 Gy) of g-ray-induced DNA
damage and its repair (during 60 min after irradiation) was
measured with the alkaline and neutral comet assay.
Comparative experiments were performed with PFGE
determining the induction of DNA-DSB by 10--50 Gy
g-irradiation and their repair during 6 h. There was no
significant difference between LCL with and without
BRCA1 mutation in any of these experiments. Therefore,
using these methods, no indication for a delayed repair of
DNA-DSB in LCL with a BRCA1 mutation was found.
However, these results do not generally exclude DNA-DSB
repair deficiency in these cell lines because the methods
applied have limited sensitivity and only measure the
speed but not the fidelity of the repair process.
Introduction
Deficiencies in DNA repair leading to chromosomal radiosensitivity i.e. enhanced induction of chromosomal aberrations and/or micronuclei by ionizing radiation has frequently
been used as a marker for cancer susceptibility. An enhanced
induction of DNA damage and impaired removal of DNA
breaks has been described in various kinds of cancer (1).
Chromosomal radiosensitivity of lymphocytes was also reported for a high percentage of patients with breast cancer and
seems to be associated in the subgroup with familial breast
cancer with the presence of a heterozygous mutation in one of
the breast cancer susceptibility genes BRCA1 or BRCA2 [for
review see (2)]. These findings are in accordance with the
central role of the BRCA gene products in maintaining
genomic stability owing to an involvement in DNA repair,
cell cycle control, chromatin remodeling and transcriptional
regulation (3--5).
Lymphoblastoid cell lines (LCL) should be a useful tool for
mechanistic studies on mutagen sensitivity owing to their
permanent growth and the availability of large pools of cells.
However, in contrast to the findings in human lymphocytes,
studies performed with LCL did not display an enhanced chromosomal mutagen sensitivity (6--8). Chromosomal aberrations
and micronuclei indicate mutations at the chromosomal level
that result mainly from misrepaired DNA double strand breaks
(DNA-DSB) (9) and deficiencies in DNA-DSB repair seem to
be involved in chromosomal radiosensitivity. To further characterize the repair capacity of LCL with a BRCA1 mutation
and to assess the usefulness of such cell lines for studying
mutagen sensitivity, we now investigated the induction and
repair of g-irradiation-induced DNA strand breaks in LCL
with and without BRCA1 mutation. We performed comparative investigations with the alkaline and the neutral version of
the comet assay (single cell gel electrophoresis) and pulsed
field gel electrophoresis (PFGE). The predominantly used
alkaline (pH 13) version of the comet assay detects a variety
of different DNA lesions, including DSB and single strand
breaks (SSB), as well as alkaline labile sites (ALS) and
incisions during DNA repair (10,11). Since ionizing radiation
induces more SSB and base damage than DSB, a specific effect
of DSB repair can be masked by the abundance of other lesions
leading to DNA migration. Therefore, we also used a ‘neutral’
(pH 8.3) version of the comet assay that has been demonstrated
to be more specific for DNA-DSB (12). Single cell gel electrophoresis under non-denaturating conditions has been shown to
be a suitable tool for studying the induction and repair of
radiation-induced DSB (12--14). Since scientific experience
with the neutral comet assay is limited and results have to be
interpreted with care, we also used PFGE, a well-established
method for the investigation of DSB induction and repair.
PFGE has been used extensively to measure DSB repair
(15--24) and a correlation between radiation sensitivity and
residual radiation-induced DNA DSB in breast cancer cells
was detected in some studies (16--21,23). Our results indicate
that the comet assay and PFGE are not suited to detect radiosensitivity in LCL with a heterozygous BRCA1 mutation and
suggest that impaired DNA-DSB repair is not a general feature
of these cell lines.
Materials and methods
Cell lines
EBV--immortalized cell lines were obtained from ATCC, Manassas, VA
(AG1011, AG09387, HCC1937BL) from Dr Thilo D€ork, Hannover, Germany
(L169, L166) and from Dr Markus Stumm, Magdeburg, Germany (L661).
L166, L661 and HCC1937BL carried a heterozygous BRCA1 mutation. L661
and L166 had a base substitution in exon 5 (T300G) and HCC1937BL carried a
single nucleotide insertion in exon 20 (5382insC). The other cell lines were
To whom correspondence should be addressed. Tel: 149 731 500 23429; Fax: 149 731 500 23438; Email: [email protected]
# The Author 2005. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.
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131
K.Trenz, P.Schütz and G.Speit
normal controls. Cells were cultured in RPMI medium (GIBCO BRL,
Eggenstein, Germany) supplemented with 15% fetal calf serum (FCS) and
antibiotics (6). All standard laboratory chemicals used in this study were
purchased from Sigma (M€
unchen, Germany).
Comet assay
Aliquots of 10 ml cell suspension (~15 000 cells) were mixed with 120 ml low
melting agarose [0.5% in phosphate-buffered saline (PBS)] and added to
microscope slides (with frosted ends), which had been covered with a bottom
layer of 1.5% agarose. In the alkaline version (pH 13), slides were processed as
described earlier (10), using a time of alkali denaturation and electrophoresis
(0.86 V/cm) of 25 min each.
The ‘neutral’ version (pH 8.3) was performed according to Wojewodzka
et al. (12). A lysis solution at pH 9.5 (2.5 M NaCl, 100 mM EDTA, 10 mM Tris,
1% Triton X-100 and 10% dimethyl sulfoxide) was used and cells were lysed
for 1 h at 4 C. A buffer with 100 mM Tris and 300 mM sodium acetate,
adjusted to pH 8.3 by HCl, was prepared for electrophoresis. The slides were
washed three times in the electrophoresis buffer, followed by an incubation for
60 min for equilibration. Slides were transferred to an electrophoresis chamber,
covered with fresh buffer and electrophoresed for 60 min at 4 C (ice bath) at
14 V (0.48 V/cm) and 60 mA. The conditions for the two comet assay versions
were settled to achieve controls with minimal DNA migration (11).
Images of 50 randomly selected ethidium bromide (10 mg/ml)-stained cells
were analysed from each coded slide. Measurements were made by image
analysis (Perceptive Instruments) determining the median tail moment
(DNA migration 3 tail intensity) of the 50 cells.
Pulsed field gel electrophoresis
PFGE was done according to Friedl et al. (25) with slight modifications. The
lymphoblastoid cells were washed in PBS and resuspended to a concentration
of 5 3 105 cells/ml. The cells were mixed with an equal volume of 1.6% LMP
agarose (InCert Agarose) at 50 C to a final concentration of 2.5 3 105 cells/ml.
The cell/agarose mixture was then transferred to plug moulds. After solidifying,
the plugs were kept in RPMI-medium (GIBCO BRL, Eggenstein, Germany) to
equilibrate for 30 min on ice. To study the induction of DSB, the cells were
irradiated in plugs with doses of 5, 10, 20, 30, 40 and 50 Gy g-irradiation
(137 Cs, Gammacell 2000, 4 Gy/min), each plug in 3 ml of RPMI-medium. After
irradiation, they were put immediately on ice to avoid repair processes and the
RPMI-medium was replaced by 5 ml 0.5 M EDTA (pH 8) for 10 min.
To study the kinetics of DSB-rejoining, seven plugs per cell line were
irradiated with 40 Gy in RPMI-medium (4 C) and immediately put on ice.
The culture medium was removed and replaced with fresh medium prewarmed
at 37 C to allow DSB repair. The cell plugs were assayed for DSB 0.5, 1, 2, 3,
4, and 6 h after irradiation. For an internal control, plugs with unirradiated cells
were also analysed for DSB at the same time points. One plug was removed at
each time point, incubated for 10 min in 0.5 M EDTA (pH 8) and then lysed for
48 h at 50 C in a lysis buffer (0.5 M EDTA, pH 8; 10 mg/ml laurylsarcosinate)
containing 1 mg/ml proteinase K. Following lysis, the plugs were washed in
rinsing buffer (10 mM Tris, 10 mM HCl, 10 mM EDTA, pH 7.5) for another
48 h at 4 C. During this time the medium was changed three times. Washed
plugs were inserted into the wells of a 0.8% agarose gel (Seakam GTG
Agarose, Sigma, M€
unchen, Germany) prepared in 0.53 TBE. PFGE was
performed using a Biometra Rotaphor (TypV) at a temperature of 22 C. The
migration conditions were set for 28 h and 1.3 V/cm with a switching time of
75 min in 2 l of 0.53 TBE buffer. After electrophoresis, the gel was stained
with ethidium bromide (1.5 mg/ml in 0.53 TBE buffer) for 1 h. The gels were
then examined under UV light, photographed and analysis was done with
Kodak digital science 1D Image Analysis Software. The percentage of DNA
migrating from the plugs into the lane (% DNA-extracted) was used as a
measure of irradiation-induced DSB (fluorescence intensity lane/fluorescence
intensity lane 1 fluorescence intensity plug). The values for the ‘real repair’
kinetics were obtained by subtracting the % DNA extracted in unirradiated
plugs from the % DNA extracted in the irradiated plugs.
Statistical analysis
All experiments were performed three times in independent tests. Differences
between mean values were tested for significance using Student’s t-test and
P 5 0.05 was considered significant.
Results
Figure 1 summarizes the g-ray-induced DNA damage in
the alkaline and the neutral version of the comet assay for the
LCL with and without a heterozygous BRCA1 mutation. All
cell lines show a dose-dependent increase in DNA migration
132
(tail moment) at doses of 2 and 5 Gy. The effects in the neutral
comet assay are generally lower and indicate some variability
among all cell lines (Figure 1A). However, in neither of the two
versions of the comet assay an obvious systematic difference
with respect to the amount of induced DNA damage could be
detected between cell lines with and without a BRCA1 mutation (Figure 1B). This means that the cell lines with a BRCA1
mutation do not reveal a clearly enhanced sensitivity towards
g-irradiation with respect to the induction of DNA strand
breaks as measured with the alkaline or neutral comet assay.
The repair kinetics of the six LCL, measured as a reduction
of induced DNA effects with time, was determined with the
alkaline and the neutral comet assay after irradiation with 2 and
5 Gy, respectively (Figures 2 and 3). Using the alkaline comet
assay, the six cell lines were irradiated with 2 Gy and the repair
kinetics were followed up to 60 min after irradiation (Figure 2).
A similar time course is observed for all LCL. LCL with a
BRCA1 mutation and control LCLs did show a continuous
decrease in the tail moment, leading to a residual effect of
520% 1 h after irradiation (Figure 2A). Two of the BRCA1
heterozygous LCL and one of the control LCL showed a slight
increase in DNA damage a few minutes after irradiation before
a reduction in the tail moment was detected. These results
indicate that LCL with a BRCA1 mutation do not generally
exhibit a reduced capacity for the repair of g-irradiationinduced DNA damage detected by the alkaline comet assay
(Figure 2B).
The repair kinetics of DNA strand breaks were then determined by the neutral comet assay (Figure 3). Owing to the
reduced sensitivity of this protocol, the initial DNA damage
was induced with 5 Gy. The removal of DNA effects appears to
be somewhat slower and the residual effects after 1 h are a little
higher compared with the alkaline version in all six LCL
(Figure 3A). The data indicate some degree of variability
between all LCL and a small increase in DNA effects 5 min
after irradiation in two control cell lines. However, a systematic difference between control LCL and LCL from women
with a BRCA1 mutation, which suggested a reduced repair
capacity in LCL with a heterozygous BRCA1 mutation, was
not detected by the neutral comet assay (Figure 3B).
PFGE was used to study the induction and repair of DNADSB in the six LCL after g-irradiation. In contrast to the
comet assay, detection of effects with PFGE required an irradiation with higher doses (10--50 Gy) and evaluation of results
is not performed on the single cell level. Figure 4 summarizes
the induction of DNA damage. A clear and dose-related
increase in DNA damage, shown as % extracted DNA from
the plug, is seen in all the six investigated cell lines. After a
dose of 50 Gy, ~60--80% of the DNA migrated outside the well,
indicating a very high amount of damaged DNA. The
dose--response curves did not reveal an enhanced sensitivity
of LCL with a BRCA1 mutation towards the induction of DSB
under these experimental conditions. The mean values even
show slightly smaller effects in BRCA1-LCL (Figure 4B).
A dose of 40 Gy was used to induce the initial DNA damage
for the determination of repair kinetics of DNA-DSB. The ‘real
DNA repair’ was followed for 6 h after irradiation, as shown in
Figure 5. The amount of extracted DNA in unirradiated cells
was subtracted from the amount of extracted DNA in cells
irradiated with 40 Gy. About 25--45% of the DNA migrated
out of the plug in the various cell lines immediately
after irradiation. All six cell lines showed a clear decrease in
DNA damage during a period of 6 h (Figure 5). Figure 5A
BRCA1 mutations and mutagen sensitivity
A 22
control
20
2 Gy
18
5 Gy
Tail moment
16
14
12
10
8
6
4
2
pH 13
B
HC L66
1
C1
93
7B
L
L1
66
L1
6
AG 9
09
38
7
AG
10
11
HC L66
1
C1
93
7B
L
L1
66
L1
6
AG 9
09
38
7
AG
10
11
0
pH 8.3
20
control
2 Gy
5 Gy
18
16
Tail moment
14
12
10
8
6
4
2
0
mean controls
mean BRCA1
pH 13
mean controls
mean BRCA1
pH 8.3
Fig. 1. Induction of DNA effects (tail moment) by 2 and 5 Gy g-irradiation in LCL from controls (L169, AG09387, AG1011) and from women
with a heterozygous BRCA1 mutation (L166, L661, HCC1937BL) determined by the alkaline comet assay at pH 13 (left) and the neutral comet assay at
pH 8.3 (right). Mean of three independent experiments, 6SEM.
indicates the high degree of variability among all cell lines with
no systematic difference between LCL with and without
BRCA1 mutation. The mean values for control LCL and LCL
with a BRCA1 mutation suggest a slightly delayed repair and
higher residual damage in the control cell lines (Figure 5B).
This should be regarded as an assay variability owing to the
limited data. There was no indication for impaired DNA-DSB
repair associated with a heterozygous BRCA1 mutation under
these experimental conditions.
Discussion
Chromosomal mutagen sensitivity has been discussed in the
context of impaired DNA-DSB repair. DNA-DSB are one of
the most relevant types of DNA damage and are directly
involved in the formation of chromosomal aberrations and
micronuclei (9). There are two distinct and complementary
mechanisms for DNA-DSB repair, homologous recombination
(HR) and non-homologous end-joining (NHEJ), to ensure the
maintenance of genome integrity in eukaryotic organisms (26).
In contrast to the HR, the NHEJ is often error-prone and small
sequence deletions are frequently introduced. Nevertheless, the
NHEJ seems to be the major pathway in adult and differentiated cells (27). In various studies, the involvement of BRCA1
in DNA repair was investigated and an involvement in HR and
NHEJ was shown (3). BRCA1 has been implicated in
homology-based repair owing to the colocalisation of BRCA1
with RAD51 in nuclear foci after irradiation (28). Furthermore,
the studies of Moynahan et al. (29,30) provide evidence for a
role of BRCA1 in the homology-directed repair. BRCA1 may
also influence NHEJ by virtue of its interaction with the
Rad50/Mre11/Nbs1 complex, which forms a foci after
133
K.Trenz, P.Schütz and G.Speit
L169
AG09387
AG1011
L166
L661
HCC1937BL
160
DNA damage [%]
140
120
100
A
L169
AG09387
AG1011
L166
L661
HCC1937BL
140
120
DNA damage [%]
A
80
60
40
20
100
80
60
40
20
0
0
10
20
30
40
50
60
0
0
time [min]
B
140
controls
B
20
30
40
time [min]
120
80
60
40
20
60
BRCA1
100
100
50
controls
BRCA1
DNA damage [%]
DNA damage [%]
120
10
80
60
40
20
0
0
10
20
30
40
time [min]
50
60
0
0
10
20
30
40
time [min]
50
60
Fig. 2. Repair kinetics of g-irradiation-induced DNA damage (2 Gy) in LCL
from controls (L169, AG09387, AG1011) and from women with a
heterozygous BRCA1 mutation (L166, L661, HCC1937BL) measured with
the alkaline comet assay (pH 13). The reduction in DNA effects (% DNA
damage) is shown for 60 min after irradiation. (A) Mean of three independent
experiments, 6SEM. (B) Mean values for the three control cell lines and the
three BRCA1 cell lines, 6SEM.
Fig. 3. Repair kinetics of g-irradiation-induced DNA damage (5 Gy) in LCL
from controls (L169, AG09387, AG1011) and from women with a
heterozygous BRCA1 mutation (L166, L661, HCC1937BL) measured with
the neutral comet assay (pH 8.3). The reduction in DNA effects (% DNA
damage) is shown for 60 min after irradiation. (A) Mean of three independent
experiments, 6SEM. (B) Mean values for the three control cell lines and the
three BRCA1 cell lines, 6SEM.
irradiation with BRCA1 (31). For the cell extract derived from
BRCA1 = mouse embryonic fibroblasts, severe defects in the
NHEJ were found (32). In addition to the defect in DSB repair,
it has been reported that both mouse BRCA1 = stem cells as
well as human BRCA1 = tumour cells, are deficient in transcription coupled repair of oxidative damage, which is also
induced by ionizing radiation (22,33,34). Recently published
studies about the BASC complex (BRCA1 associated genome
surveillance complex) suggest a coordinating function of
BRCA1 in a variety of different DNA repair pathways. In the
BASC-complex, DNA damage repair proteins such as MSH2,
MSH6 and MLH1 as well as ATM, NBS1, MRE11 and BLM
are included (35,36).
In contrast to lymphocytes, LCL from breast cancer patients
with a BRCA1 mutation did not exhibit chromosomal radiosensitivity (6). This finding was recently confirmed in a study
using blood and LCL from patients with breast cancer (8). LCL
established from patients with breast cancer who showed an
elevated chromosomal radiosensitivity in fresh blood samples
did not reveal this phenomenon. On the other hand, impaired
fidelity of DNA-DSB end-joining was reported for LCL with
various types of BRCA1 mutations (37,38). We therefore performed experiments with assays for the detection of DNA-DSB
to further elucidate the relationship between BRCA1 mutations, chromosomal radiosensitivity and DNA-DSB repair in
LCL. The comet assay has already been used to study mutagen
sensitivity in patients with breast cancer but conflicting results
have been published (6,24,39--43). In general, the comet assay
should be well suited for these types of investigations, because
it follows the induction and removal of DNA damage with high
sensitivity (10). However, if chromosomal mutagen sensitivity
is based mainly on impaired DNA-DSB repair, the abundance
of SSB and other types of DNA lesions also leading to DNA
migration in the alkaline version of the comet assay might
mask a specific difference in DSB repair. Therefore, the neutral
version of the comet assay, which is more specific for DNADSB, might be the better option to detect differences in DSB
repair and radiosensitivity (17,20). The protocol for the neutral
comet assay used here has been established previously and
tested for its specificity for DNA-DSB (12). Our results indicate that cells with one mutant BRCA1 allele rejoin DNA-SSB
and DNA-DSB to an extent similar to that observed in LCL
134
BRCA1 mutations and mutagen sensitivity
120
100
% extracted DNA
A
AG1011
AG09387
L169
HCC1937BL
L166B
L661
80
AG1011
60
AG09387
L169
HCC1937BL
50
L166B
% extracted DNA
A
60
40
L661
30
20
40
10
20
0
0
0
0
B
10
20
30
irradiation dose [Gy]
40
2
4
50
B
25
controls
45
BRCA
control
40
20
BRCA1
% extracted DNA
DN
35
% extracted DNA
6
time [h]
30
25
20
15
15
10
5
10
0
5
0
2
4
6
time [h]
0
0
10
20
30
irradiation dose [Gy]
40
50
Fig. 4. Induction of DNA damage by g-irradiation (10--50 Gy) measured
by PFGE in LCL from controls (L169, AG09387, AG1011) and women with a
BRCA1 mutation (L169, AG09387, AG1011). (A) Mean of three
independent experiments, 6SEM. (B) Mean values for the three control cell
lines and the three BRCA1 cell lines, 6SEM.
with two functional alleles. The slight increase observed at the
beginning of the repair period in some of the cell lines might
indicate that the effect of ongoing repair is stronger than the
elimination of lesions leading to DNA migration. It cannot be
excluded that the optimal (early) time point to detect differences in DSB repair was missed. This should be considered in
future studies and a modification of the comet assay with
irradiation of cells already embedded on the slides (20) might
be useful. At present, our results indicate that neither the
alkaline version of the comet assay nor the neutral version is
well suited to clearly distinguish between LCL with and without BRCA1 mutation.
The sensitivity of the comet assay for the detection of
reduced DSB repair capacities still has to be defined. A recent
study reported that the time course of human replication protein A (RPA) and gamma-H2AX foci association correlated
Fig. 5. Repair kinetics of g-irradiation-induced DNA damage (40 Gy)
measured by PFGE in LCL from controls (L169, AG09387, AG1011) and
from women with a heterozygous BRCA1 mutation (L166, L661,
HCC1937BL). Reduction in DNA damage (% DNA extracted from the plug)
is shown for 6 h after irradiation. (A) Mean of three independent experiments,
6SEM. (B) Mean values for the three control cell lines and the three
BRCA1 cell lines, 6SEM.
well with the DSB repair activity detected by the neutral comet
assay. Additionally, radiosensitive ataxia telangiectasia (AT)
cells were found to be deficient in RPA and gamma-H2AX
colocalisation after irradiation (44). However, the comet assay
was not able to detect the reduced repair capacity in homozygous and heterozygous AT-LCL (45,46) despite their radiosensitivity in the micronucleus test (46,47).
Our negative results obtained with the neutral comet assay
are in accordance with the results from the PFGE experiments.
There was some variability between cell lines but no fundamental difference associated with the presence of a BRCA1
mutation. In contrast to the comet assay, DSB are induced
using a much higher dose of g-irradiation and repair was
followed for a much longer period of time. Although PFGE is
a well-established method for the evaluation of DSB repair,
it is rather insensitive and the experimental conditions used in
135
K.Trenz, P.Schütz and G.Speit
the comet assay are more closely related to the cytogenetic
tests for chromosomal mutagen sensitivity. Our PFGE results
are in agreement with the study of Nieuwenhuis et al. (24) who
investigated lymphocytes and fibroblasts from 18 BRCA1 and
6 BRCA2 heterozygous subjects and four control subjects. A
connection between impaired DNA repair capacity and mutations in BRCA1 and BRCA2 genes could not be established.
When the repair in fibroblast lines was analysed by PFGE, the
variation between fibroblasts in the BRCA1 1= group was
greater compared with the two other groups, but no indication
of an altered DSB rejoining capacity was apparent (24). A
BRCA1-deficient tumour cell line with obvious radiation sensitivity in the colony forming assay repaired most of the
radiation-induced DNA damage within 2 h after radiation without any indication for delayed repair (22). The proficiency of
DSB repair was confirmed in this cell line in a later study (35).
A few studies using PFGE describe that BRCA1 protects cells
against radiation-induced DSB and contributes to DSB repair
as indicated by delayed or impaired repair kinetics of radiationinduced damage. This was first shown for a BRCA1-deficient
human tumour cell line, which failed to repair DSB efficiently
(21). In contrast to our results, some LCL heterozygous for
BRCA1 and BRCA2 did show a higher amount of residual
DSB after irradiation (23). These authors investigated two
BRCA1 cell lines and three BRCA2 cell lines in comparison
with two control LCL. Approximately 50% of the induced
damage was repaired within the first hour, irrespective of the
genotype. Differences appeared up to 6 h with clear deficiencies in repair capacity in BRCA1 and BRCA2 heterozygous
cell lines. The discrepancy between these results and our study
cannot be explained at present, but it should be stressed that
both studies are based on a small number of cell lines and the
assay variability of PFGE is considerable. The sensitivity of
PFGE for the detection of deficient repair of DSB may also
depend on the experimental conditions used. It has been shown
that repair deficiency is detected by PFGE in fibroblasts from
AT patients and from obligate AT heterozygotes only after low
dose-rate g-irradiation and is due to a deficiency in the repair
of a small fraction of DSB (48). Possibly, the unequivocal
detection of radiosensitivity in cells with a BRCA1 mutation
requires a refinement of the PFGE technique (17).
In conclusion, this study indicates that LCL with a heterozygous BRCA1 mutation that did not show chromosomal
radiosensitivity in the micronucleus test in contrast to lymphocytes with the same mutation, also did not reveal a repair
deficiency for DNA-DSB when tested with the neutral comet
assay and PFGE. Our results contrast with the recent findings
for some other LCL with a heterozygous mutation in BRCA1,
suggesting that the fidelity of DNA end-joining is strongly
reduced (37,38). However, it is not yet known whether these
cell lines exhibit increased chromosomal radiosensitivity or
whether these two effects are unrelated.
Acknowledgements
We wish to thank Dr A.Friedl, M€unchen and Dr E.Vock, Biberach, for valuable
suggestions for the use of PFGE. This study was financially supported by a
grant from the Deutsche Forschungsgemeinschaft (SP 274/8-1).
References
1. Berwick,M. and Vineis,P. (2000) Markers of DNA repair and susceptibility to cancer in humans: an epidemiologic review. J. Natl Cancer Inst.,
92, 874--897.
136
2. Speit,G. and Trenz,K. (2004) Chromosomal mutagen sensitivity
associated with mutations in BRCA genes. Cytogenet Genome Res.,
104, 325--332.
3. Venkitaraman,A.R. (2002) Cancer susceptibility and the functions of
BRCA1 and BRCA2. Cell, 108, 171--182.
4. Welcsh,P.L., Owens,K.N. and King,M.C. (2000) Insights into the
functions of BRCA1 and BRCA2. Trends Genet., 16, 69--74.
5. Deng,C.X. and Brodie,S.G. (2000) Roles of BRCA1 and its interacting
proteins. Bioessays, 22, 728--737.
6. Trenz,K., Landgraf,J. and Speit,G. (2003) Mutagen sensitivity of human
lymphoblastoid cells with a BRCA1 mutation. Breast Cancer Res. Treat.,
78, 69--79.
7. Baeyens,A., Thierens,H., Claes,K., Poppe,B., Messiaen,L., De Ridder,L.
and Vral,A. (2002) Chromosomal radiosensitivity in breast cancer patients
with a known or putative genetic predisposition. Br. J. Cancer, 87,
1379--1385.
8. Baeyens,A., Thierens,H., Vandenbulcke,K., De Ridder,K. and Vral,A.
(2004) The use of EBV-transformed cell lines of breast cancer patients to
measure chromosomal radiosensitivity. Mutagenesis, 19, 285--290.
9. Obe,G., Pfeiffer,P., Savage,J.R., Johannes,C., Goedecke,W., Jeppesen,P.,
Natarajan,A.T., Martinez-Lopez,W., Folle,G.A. and Drets,M.E. (2002)
Chromosomal aberrations: formation, identification and distribution.
Mutat. Res., 504, 17--36.
10. Speit,G. and Hartmann,A. (1999) The comet assay (single-cell gel test). A
sensitive genotoxicity test for the detection of DNA damage and repair.
Methods Mol. Biol., 113, 203--212.
11. Tice,R.R., Agurell,E., Anderson,D., Burlinson,B., Hartmann,A.,
Kobayashi,H., Miyamae,Y., Rojas,E., Ryu,J.C. and Sasaki,Y.F. (2000)
Single cell gel/comet assay: guidelines for in vitro and in vivo genetic
toxicology testing. Environ. Mol. Mutagen., 35, 206--221.
12. Wojewodzka,M., Buraczewska,I. and Kruszewski,M. (2002) A modified
neutral comet assay: elimination of lysis at high temperature and
validation of the assay with anti-single-stranded DNA antibody. Mutat.
Res., 518, 9--20.
13. Olive,P.L., Wlodek,D. and Banath,J.P. (1991) DNA double-strand breaks
measured in individual cells subjected to gel electrophoresis. Cancer Res.,
51, 4671--4676.
14. Singh,N.P. and Stephens,R.E. (1997) Microgel electrophoresis:
sensitivity, mechanisms, and DNA electrostretching. Mutat. Res., 383,
167--175.
15. Woudstra,E.C., Driessen,C., Konings,A.W. and Kampinga,H.H. (1998)
DNA damage induction and tumour cell radiosensitivity: PFGE and halo
measurements. Int. J. Radiat. Biol., 73, 495--502.
16. Nunez,M.I., Guerrero,M.R., Lopez,E., del Moral,M.R., Valenzuela,M.T.,
Siles,E., Villalobos,M., Pedraza,V., Peacock,J.H. and Ruiz de
Almodovar,J.M. (1998) DNA damage and prediction of radiation response
in lymphocytes and epidermal skin human cells. Int. J. Cancer, 76,
354--361.
17. Sarkaria,J.N., Bush,C., Eady,J.J., Peacock,J.H., Steel,G.G. and
Yarnold,J.R. (1998) Comparison between pulsed-field gel electrophoresis
and the comet assay as predictive assays for radiosensitivity in fibroblasts.
Radiat. Res., 150, 17--22.
18. Kiltie,A.E., Ryan,A.J., Swindell,R., Barber,J.B., West,C.M., Magee,B.
and Hendry,J.H. (1999) A correlation between residual radiation-induced
DNA double-strand breaks in cultured fibroblasts and late radiotherapy
reactions in breast cancer patients. Radiother. Oncol., 51, 55--65.
19. Badie,C., Iliakis,G., Foray,N., Alsbeih,G., Cedervall,B., Chavaudra,N.,
Pantelias,G., Arlett,C. and Malaise,E.P. (1995) Induction and rejoining of
DNA double-strand breaks and interphase chromosome breaks after
exposure to X rays in one normal and two hypersensitive human fibroblast
cell lines. Radiat. Res., 144, 26--35.
20. Price,M.E., McKelvey-Martin,V.J., Robson,T., Hirst,D.G. and
McKeown,S.R. (2000) Induction and rejoining of DNA double-strand
breaks in bladder tumor cells. Radiat. Res., 153, 788--794.
21. Scully,R., Ganesan,S., Vlasakova,K., Chen,J., Socolovsky,M. and
Livingston,D.M. (1999) Genetic analysis of BRCA1 function in a defined
tumor cell line. Mol. Cell, 4, 1093--1099.
22. Abbott,D.W., Thompson,M.E., Robinson-Benion,C., Tomlinson,G.,
Jensen,R.A. and Holt,J.T. (1999) BRCA1 expression restores radiation
resistance in BRCA1-defective cancer cells through enhancement of
transcription-coupled DNA repair. J. Biol. Chem., 274, 18808--18812.
23. Foray,N., Randrianarison,V., Marot,D., Perricaudet,M., Lenoir,G. and
Feunteun,J. (1999) Gamma-ray-induced death of human cells carrying
mutations of BRCA1 or BRCA2. Oncogene, 18, 7334--7342.
24. Nieuwenhuis,B., Assen-Bolt,A.J., Waarde-Verhagen,M.A., Sijmons,R.H.,
Van der Hout,A.H., Bauch,T., Streffer,C. and Kampinga,H.H. (2002)
BRCA1 mutations and mutagen sensitivity
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
BRCA1 and BRCA2 heterozygosity and repair of X-ray-induced DNA
damage. Int. J. Radiat. Biol., 78, 285--295.
Friedl,A.A., Kraxenberger,A. and Eckardt-Schupp,F. (1995) An electrophoretic approach to the assessment of the spatial distribution of
DNA double-strand breaks in mammalian cells. Electrophoresis, 16,
1865--1874.
Khanna,K.K. and Jackson,S.P. (2001) DNA double-strand breaks:
signaling, repair and the cancer connection. Nat. Genet., 27, 247--254.
Hoeijmakers,J.H. (2001) Genome maintenance mechanisms for preventing
cancer. Nature, 411, 366--374.
Scully,R., Chen,J., Ochs,R.L., Keegan,K., Hoekstra,M., Feunteun,J. and
Livingston,D.M. (1997) Dynamic changes of BRCA1 subnuclear location
and phosphorylation state are initiated by DNA damage. Cell, 90,
425--435.
Moynahan,M.E., Chiu,J.W., Koller,B.H. and Jasin,M. (1999) Brca1
controls homology-directed DNA repair. Mol. Cell, 4, 511--518.
Moynahan,M.E., Cui,T.Y. and Jasin,M. (2001) Homology-directed DNA
repair, mitomycin-c resistance, and chromosome stability is restored with
correction of a Brca1 mutation. Cancer Res., 61, 4842--4850.
Zhong,Q., Chen,C.F., Li,S., Chen,Y., Wang,C.C., Xiao,J., Chen,P.L.,
Sharp,Z.D. and Lee,W.H. (1999) Association of BRCA1 with the hRad50hMre11-p95 complex and the DNA damage response. Science, 285,
747--750.
Zhong,Q., Boyer,T.G., Chen,P.L. and Lee,W.H. (2002) Deficient
nonhomologous end-joining activity in cell-free extracts from Brca1-null
fibroblasts. Cancer Res., 62, 3966--3970.
Gowen,L.C., Avrutskaya,A.V., Latour,A.M., Koller,B.H. and Leadon,S.A.
(1998) BRCA1 required for transcription-coupled repair of oxidative DNA
damage. Science, 281, 1009--1012.
Le Page,F., Randrianarison,V., Marot,D., Cabannes,J., Perricaudet,M.,
Feunteun,J. and Sarasin,A. (2000) BRCA1 and BRCA2 are necessary for
the transcription-coupled repair of the oxidative 8-oxoguanine lesion in
human cells. Cancer Res., 60, 5548--5552.
Wang,Y., Cortez,D. Yazdi,P., Neff,N., Elledge,S.J. and Qin,J. (2000)
BASC, a super complex of BRCA1-associated proteins involved in the
recognition and repair of aberrant DNA structures. Genes Dev., 14,
927--939.
Futaki,M. and Liu,J.M. (2001) Chromosomal breakage syndromes and the
BRCA1 genome surveillance complex. Trends Mol. Med., 7, 560--565.
Baldeyron,C., Jacquemin,E., Smith,J., Jacquemont,C., De Oliveira,I.,
Gad,S., Feunteun,J., Stoppa-Lyonnet,D. and Papadopoulo,D. (2002) A
single mutated BRCA1 allele leads to impaired fidelity of double strand
break end-joining. Oncogene, 21, 1401--1410.
Coupier,I., Baldeyron,C., Rousseau,A., Mosseri,V., Pages-Berhouet,S.,
Caux-Moncoutier,V., Papadopoulo,D. and Stoppa-Lyonnet,D. (2004)
Fidelity of DNA double-strand break repair in heterozygous cell lines
harbouring BRCA1 missense mutations. Oncogene, 23, 914--919.
Jaloszynski,P., Kujawski,M., Czub-Swierczek,M., Markowska,J. and
Szyfter,K. (1997) Bleomycin-induced DNA damage and its removal in
lymphocytes of breast cancer patients studied by comet assay. Mutat. Res.,
385, 223--233.
Alapetite,C., Thirion,P., de la Rochefordiere,A., Cosset,J.M. and
Moustacchi,E. (1999) Analysis by alkaline comet assay of cancer patients
with severe reactions to radiotherapy: defective rejoining of radioinduced
DNA strand breaks in lymphocytes of breast cancer patients. Int. J.
Cancer, 83, 83--90.
Rothfuss,A., Schutz,P., Bochum,S., Volm,T., Eberhardt,E., Kreienberg,R.,
Vogel,W. and Speit,G. (2000) Induced micronucleus frequencies in
peripheral lymphocytes as a screening test for carriers of a BRCA1
mutation in breast cancer families. Cancer Res., 60, 390--394.
Rajeswari,N., Ahuja,Y.R., Malini,U., Chandrashekar,S., Balakrishna,N.,
Rao,K.V. and Khar,A. (2000) Risk assessment in first degree female
relatives of breast cancer patients using the alkaline Comet assay.
Carcinogenesis, 21, 557--561.
Smith,T.R., Miller,M.S., Lohman,K.K., Case,L.D. and Hu,J.J. (2003)
DNA damage and breast cancer risk. Carcinogenesis, 24, 883--889.
Balajee,A.S. and Geard,C.R. (2004) Replication protein A and gammaH2AX foci assembly is triggered by cellular response to DNA doublestrand breaks. Exp. Cell Res., 300, 320--334.
Humar,B., Muller,H. and Scott,R.J. (1997) Elevated frequencies of p53independent apoptosis after irradiation increases levels of DNA breaks in
ataxia telangiectasia lymphoblasts. Int. J. Radiat. Biol., 72, 257--269.
Speit,G., Trenz,K., Sch€utz,P., Bendix,R. and D€ork,T. (2000) Mutagen
sensitivity of human lymphoblastoid cells with a BRCA1 mutation in
comparison to ataxia telangiectasia heterozygote cells. Cytogenet. Cell
Genet., 91, 261--266.
47. Jaworska,A., Wojewodzka,M. and De Angelis,P. (2002) Radiation
sensitivity and the status of some radiation sensitivity markers
in relatively sensitive lymphoid cells. Radiat. Biol. Radioecol., 42,
595--599.
48. Blocher,D., Sigut,D. and Hannan,M.A. (1991) Fibroblasts from ataxia
telangiectasia (AT) and AT heterozygotes show an enhanced level of
residual DNA double-strand breaks after low dose-rate gamma irradiation
as assays by pulsed field gel electrophoresis. Int. J. Radiat. Biol., 60,
791--802.
Received on December 16, 2004; revised on February 9, 2005;
accepted on February 21, 2005
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