Cancer cells exhibit characteristics that distinguish them from their normal counterparts.
Three cellular functions tend to be inappropriately regulated in a neoplasm. First,
the normal constraints on cellular proliferation are relaxed. Second, differentiation
can be distorted. Third, chromosomal and genetic organisation may be destabilised
such that variant cells arise with high frequency. All those factors do not only determine
the growth and malignant characteristics of the tumour but also their responsiveness
to radiation. This ‘intrinsic’ radiosensitivity differs largely between tumour types
and is at least partly due to the different sensitivity of the respective tumour cells
Deacon et al, 1984; Fertil and Malaise, 1985). It was further shown that within one
type of tumours, the outcome of the individual patients after radiotherapy was reflected
by the in vitro radiosensitivity (SF2) of their tumour cells (West et al, 1993, 1995;
Stausbol-Gron and Overgaard, 1999; Björk-Eriksson et al, 2000).
It is generally accepted that among the DNA damage induced mainly double-strand breaks
(dsb) (Bryant, 1984; Ward, 1988; Wurm et al, 1994; Dikomey et al, 1998; Pfeiffer et
al, 2000), and particularly residual dsb (Dikomey et al, 1998, 2000) are responsible
for cell killing by ionising radiation. The number of residual dsb depends on both,
the number of dsb induced and on the respective repair capacity. In normal cells,
no variation was found for the number of induced dsb (Dikomey et al, 1998, 2000),
illustrating that differences are only due to variations in the repair capacity.
This picture appears to be much more complex and even contradictory for tumour cells.
11 out of 13 studies reported on a variation in the number of induced dsb (Kelland
et al, 1988; Schwartz et al, 1988, 1990, 1991; McMillan et al, 1990; Giaccia et al,
1992; Olive et al, 1994; Ruiz de Almodovar et al, 1994; Zaffaroni et al, 1994; McKay
and Kefford, 1995; Whitaker et al, 1995; Woudstra et al, 1998; Eastham et al, 2001),
but only three found a correlation with cell killing (McMillan et al, 1990; Ruiz de
Almodovar et al, 1994; Whitaker et al, 1995). In repair studies, a correlation between
cell killing and the number of dsb was only found after short repair intervals (up
to 2 h) (Schwartz et al, 1988, 1990; Giaccia et al, 1992; Zaffaroni et al, 1994),
but never for nonreparable dsb.
These heterogeneous results may be partly explained by the techniques used (see also
McMillan et al, 2001). Several studies raised doubts as to whether neutral filter
elution exclusively detects dsb (Hutchinson, 1989; Okayasu and Iliakis, 1989; Wlodek
and Olive, 1990). Pulsed-field gel electrophoresis (PFGE), currently most widely used,
has the advantage to resolve large DNA fragments according to the molecular size permitting,
in principle, a direct quantification of dsb. However, the accurate analysis of the
profile of the continuous fragment distribution is not trivial (Ager et al, 1990;
Kraxenberger et al, 1994; Cedervall et al, 1995). In addition, PFGE could be complicated
by paradoxical migration patterns (Carle et al, 1986; Chu, 1991; Löbrich et al, 1993).
Therefore, PFGE and constant-field gel electrophoresis (CFGE) are preferably used
to quantify only the fraction of DNA fragments released (FDR) from the bulk DNA (Blöcher
et al, 1989; Stamato and Denko, 1990; Iliakis et al, 1991a, 1991b). All three methods
are sensitive to replication (Okayasu et al, 1988; Stamato and Denko, 1990; Iliakis
et al, 1991a, 1991b; Dahm-Daphi and Dikomey, 1995), which might be important for continuously
proliferating tumour cells.
To this end, we introduced a modification of the standard electrophoresis termed ‘graded-field
gel electrophoresis’ (GFGE) (Dahm-Daphi and Dikomey, 1995, 1996; Zhou et al, 1997a,
1997b). GFGE operates at stepwise increased electric field strength resulting in distinct
bands that contain fragments of different molecular weights. The analysis then permits
direct calculation of the number of dsb independent of the cell cycle distribution.
Here we applied the GFGE to determine the number of dsb induced in nine tumour cell
lines and for control in seven normal fibroblast lines and compared it with the respective
data obtained by CFGE. We further measured the kinetics of dsb repair and the residual
damage 24 h after irradiation in order to clarify, whether dsb critically determine
tumour cell survival.
MATERIALS AND METHODS
Cell lines and culture conditions
The LNCaP and DU145 cell lines were originally isolated from metastatic lesions of
patients suffering from prostate cancer (Stone et al, 1978; Horoszewicz et al, 1983)
and purchased from DSMZ (Braunschweig, Germany) as American Type Culture Collection
(ATCC) cell lines. HeLa cells were derived from a cervical carcinoma (Scherer et al,
1953) obtained from Dr Aubee. The T47D-B8 and MCF-7 cell lines descended from human
breast carcinomas (Soule et al, 1973; Soto et al, 1986; Ruiz de Almodovar et al, 1994).
The MCF-7 subclones BB and Bus were described to differ from MCF-7 parental cells
with respect to dsb induction and repair, p53 status, cell cycle and apoptosis (Nunez
et al, 1995; Siles et al, 1995). These cells as well as the RT112 bladder carcinoma
cell line (Masters et al, 1986) were gifted by Dr Ruiz de Almodevar. SCC4451 cells
were established from a squamous cell carcinoma of the head and neck obtained from
Dr Zölzer (Zölzer et al, 1995). All cells were either grown in RMPI 1640 or DMEM (Life
Science Technology/BRL, Karlsruhe, Germany) supplemented with 10% FCS and penicillin/streptomycin
in 5% CO2.
Fibroblast cells, HF-1, -2, -7, -8, -46, -60 and HF, were established from punch biopsies
as described elsewhere (Borgmann et al, 2002). CHO cells were kept in α-MEM medium.
The experiments were performed with nearly confluent tumour cells and confluent fibroblasts.
Clonogenic cell survival
Tumour cell survival was assessed by colony formation assay. Near-confluent cultures
were X-irradiated at 220 kVp, at a dose-rate of 2 Gy min−1. Irradiated cells were
immediately plated and grown for 2–3 weeks with one medium change. Stained colonies
of more than 50 cells were counted. Experiments were repeated thrice with three replicates
each.
Constant-field gel electrophoresis
Subconfluent cells were suspended (3 × 106 ml in 0.8% low melting point agarose (Bio-Rad,
Munich, Germany) and pipetted into 180 μl plug moulds to solidify (Dahm-Daphi and
Dikomey, 1995). Those agarose plugs were irradiated on ice to prevent DNA repair during
irradiation and thereafter directly subjected to cell lysis (0.4 M EDTA, 2% N-lauryl
sarcosine, and 1 mg ml−1 proteinase K, all Sigma, Deisenhofen, Germany). Lysis was
started on ice for 30 min and continued at 37°C overnight. The plugs were washed thrice
with Tris-EDTA buffer and sliced into pieces containing about 105 cells, which were
inserted into a 14 × 20 cm 0.8% agarose gel (High-grade ultrapure, Bio-Rad). The gel
was then covered with a thin overlayer of 0.8% agarose to avoid light fraction artefacts
upon optical imaging. Electrophoresis was performed at 0.6 V cm−1 for 30 h in 0.5
× TBE buffer (45 mM Tris base, 45 mM boric acid, 2 mM EDTA) in a conventional apparatus
(Subcell, Bio-Rad). The gel was then stained overnight (0.5 μg ml−1 ethidium bromide),
destained overnight in ddH2O and CCD camera (Sony XC-75CE) equipped with an image
analysis system (Optimas, Silverspring, MD, USA) was used to quantify the FDR.
Graded-field gel electrophoresis
Cells were treated as before but electrophoresis was performed differently. Running
conditions were now 0.6 V cm−1 for 30 h followed by 1.5 V cm−1 for 6 h. Compared to
the regimen published previously (Dahm-Daphi and Dikomey, 1995) the present protocol
was restricted to two different field strengths sufficient for the current purpose.
Detailed analysis and mathematics were described elsewhere (Dahm-Daphi and Dikomey,
1995). In principle, analysis was based on an equation given by Blöcher (1990) describing
the sigmoid curve of the total FDR obtained by PFGE. Here, the total FDR corresponds
to the sum of the FDR of the two bands (GFGE at 0.6 and 1.5 V cm−1, Figure 2). The
curves of total FDR and FDR at 1.5 V cm−1 were both sigmoid (Figure 4) and could,
hence, be fitted by the equation described by Blöcher (1990). The third curve (band
1) corresponds to the difference between both fits. The χ
2 fits were calculated simultaneously and gave the number of dsb in unirradiated cells,
of dsb induced per DNA-unit, the maximum DNA fragment size in each band and the factor
of retention (f
ret).
Cell cycle analysis
Cell cycle distribution of PI-stained cells was determined by flow cytometry in an
FACScan (Becton Dickenson, Heidelberg, Germany).
Statistics
Each experiment was repeated three times and the data were given as a mean with its
standard error (±s.e.m.). Statistical analysis, data fitting and graphics were performed
by means of the Prism 3.1 computer program (GraphPad Software, San Diego, USA).
RESULTS
The nine tumour cell lines showed a wide range of radiosensitivity (Figure 1
Figure 1
Cellular radiosensitivity of human tumour cell lines. Cell survival was determined
by means of colony formation assay. Data were fitted to the linear-quadratic equation
−ln(S/S
0)=αD+βD
2 where S is the surviving fraction, S
0 the plating efficiency of unirradiated cells and D the X-ray dose.
). The SF2, the linear-quadratic parameters α and β and the mean inactivation dose,
D
bar (Fertil et al, 1984), were calculated (Table 1
Table 1
Parameters of cellular radiosensitivity and cell cycle distribution
0 Gy
150 Gy
Cell lines
SF2a
α (Gy−1)b
β (Gy−2)b
D
bar (Gy)c
Gd
1
S
G2/M
G1
S
G2/M
RT112
0.62±0.02
0.18±0.06
0.028±0.007
3.2±0.5
55
33
13
43
54
2
DU145
0.63±0.05
0.12±0.02
0.055±0.005
2.85±0.3
54
36
10
47
44
9
SSC4451
0.41±0.07
0.38±0.15
0.032±0.018
2.04±0.5
80
11
9
83
10
7
HeLa
0.43±0.03
0.33±0.06
0.043±0.007
2.1±0.2
70
18
12
43
40
17
LNCaP
0.17±0.01
0.87±0.06
0.014±0.011
1.11±0.1
78
15
7
68
23
9
T47D-B8
0.46±0.06
0.29±0.4
0.045±0.006
2.21±0.2
65
26
9
53
14
33
MCF-7
0.53±0.06
0.16±0.04
0.078±0.006
2.35±0.3
73
16
11
60
25
15
MCF7-BB
0.51±0.05
0.28±0.03
0.027±0.005
2.55±0.1
47
43
10
70
16
14
MCF7-Bus
0.31±0.03
0.52±0.03
0.03±0.005
1.64±0.1
76
14
10
52
3
13
a
Surviving fraction for a X-ray dose of 2 Gy.
b
Linear and quadratic term of the dose response.
c
Dbar, mean inactivation dose.
d
Percentage of cells in G1, S or G2M-phase 24 h after 0 or 150 Gy.
). The D
bar of the most radiosensitive cell line (LNCaP) was 2.9 times lower as compared to
the most radioresistant strain (RT112).
Induction of dsbs
Figure 2
Figure 2
Constant- and graded-field gel electrophoresis, CFGE (upper) and GFGE (lower). CFGE
was run at 0.6 V cm−1 for 30 h and GFGE at two field strengths of 0.6 V cm−1 for 30 h
followed by 1.5 V cm−1 for 6 h. The fluorescence intensity of each band in each lane
was recorded by a CCD video camera.
shows dose-dependent separation of radiation-induced DNA fragments for MCF7-BB cells
using CFGE (A) and GFGE (B). CFGE was run at a constant field strength of 0.6 V cm−1
collecting all released DNA fragments in a single band. The FDR, as determined by
CFGE for all tumour cell lines (Figure 3
Figure 3
(A) Induction of dsb measured by CFGE. Cells were irradiated on ice with doses up
to 140 Gy immediately followed by the measurement of dsb by CFGE. The increase of
FDR with dose was fitted by nonlinear regression. The initial slope of FDR was determined
by linear regression of the data obtained for doses up to 20 Gy (insrt). (B) Correlation
between the number of dsb induced, as calculated from Figure 4, and the initial slope,
as taken from Figure 3A. The symbols correspond to those in Figure 1.
), was found to increase with dose finally reaching a plateau at doses of 100–140 Gy.
Clear differences were obvious for both the initial slope (see inset) and the final
plateau. The initial slope varied by a factor of 2 (Table 2
Table 2
Parameters of initial and residual dsb for tumour and normal cells
Cell lines
Initial slopea (Gy−1)
k
b 10−12 (Gy−1 Da−1)
f
ret
c (%)
N24 h(150 Gy)d (Gy-equival.)
Tumour cell lines
RT112
0.010±0.001
5.75±0.77
33±3
37.5±4.9
DU145
0.012±0.001
6.39±0.64
30±4
24.1±4.2
SSC4451
0.018±0.001
8.18±0.63
23±4
9.9±2.4
HeLa
0.021±0.003
8.31±0.72
11±3
>150
LNCaP
0.020±0.001
8.31±0.76
12±2
10.2±2.5
T47D-B8
0.018±0.001
9.71±0.70
28±4
>150
MCF-7
0.017±0.002
8.43±0.71
16±3
12.8±1.8
MCF7-BB
0.018±0.001
7.92±0.59
19±3
14.5±2.3
MCF7-Bus
0.020±0.002
11.00±0.65
40±5
>150
Mean
8.22±1.6
Normal human Fibroblasts
HF-1
Nde
7.76±0.71
3±2
Nd
HF-46
Nd
7.80±0.63
22±3
Nd
HF-2
Nd
8.43±0.69
15±3
Nd
HF-60
Nd
8.05±0.72
17±2
Nd
HF-F
Nd
8.69±0.68
20±5
Nd
HF-8
Nd
7.16±0.75
6±3
Nd
HF-7
Nd
7.54±0.60
6±3
Nd
Mean
7.9±0.50
a
Initial slope calculated by linear regression from data shown in the inset of Figure
3.
b
Number of dsb per Gy and per Da calculated from the data plotted in Figure 4.
c
Fraction of fragments that did not migrate to band 1 because of being retained in
the plug.
d
Number of dsb measured 24 h after irradiation with 150 Gy expressed in Gy-equivalents.
e
Not determined.
, 1st row) and the final plateau by 1.6.
These data suggested that the nine tumour strains studied differ in the number of
dsb induced. However, the absolute number cannot be deduced from these data. Therefore,
induction was also measured by GFGE, as previously described (Dahm-Daphi and Dikomey,
1995). Electrophoresis was run at two field strengths, which resulted in two distinct
bands containing DNA fragments of different molecular weights (Figure 2B). GFGE hence
allows to calculate the absolute number of dsb induced in each cell line. Fractions
of DNA released (FDR) to bands 1 and 2 together with the total DNA released were measured
for all nine tumour and also for seven human fibroblast lines (Figure 4
Figure 4
Induction of dsb measured by GFGE. Cells were irradiated on ice with doses up to 140 Gy
immediately followed by the measurement of dsb by GFGE. GFGE was run for nine tumour
and seven primary fibroblast strains. For each cell line, the FDR of band 1 (circles),
band 2 (triangles) and the sum of both (squares) were commonly fitted by nonlinear
regression (see text and Dahm-Daphi and Dikomey, 1995).
). In all cases, FDR of band 1 first increased with the dose, reaching a maximum at
a certain dose and declined thereafter. In contrast, band 2 increased continuously.
The fraction of total DNA released, which is the sum of FDR in bands 1 and 2, gave
a similar curve to that of CFGE (Figure 3). Bands 1 and 2 differed considerably between
the cell lines, which was most obvious for the dose at which band 1 reached its maximum.
For example, RT112 and DU145 cells band 1 reached this maximum at doses of 55–60 Gy,
in contrast to a two-fold lower dose of 30 Gy measured for MCF-7-Bus cells.
For each strain, the FDR in bands 1 and 2 were simultaneously fitted as function of
dose using a model previously described in detail (Dahm-Daphi and Dikomey, 1995).
Those fits gave the number of dsb and a retention factor (f
ret). The comparison of all strains gave by fitting the size of the fragments in each
band. According to these fits band 1 contained DNA fragments of 5.25–11.0 Mbp and
band 2 of less than 5.25 Mbp. Theses values are slightly higher than previously reported
(Dahm-Daphi and Dikomey, 1995). Now, 16 different cell lines have been analysed instead
of only two data sets before (Dahm-Daphi and Dikomey, 1995). The number of dsb induced
per Gy and Da was found to vary by a factor of 2 between 5.75 for RT112 and 11.5 ×
10−12 dsb/Gy/Da for MCF7-Bus (Table 2). The respective range for the seven normal
fibroblast lines was significantly smaller (7.2–8.6 × 10−12 dsb/Gy/Da). These values
are equivalent to a variation of 14–27 dsb/Gy/diploid DNA content for tumour cells
and 18–20 dsb/Gy/diploid DNA content for normal fibroblasts.
Figure 3B shows the relationship between the initial slope of the FDR curves measured
by CFGE (Figure 3A) and the number of induced dsb as obtained from GFGE (Figure 4).
There was a significant correlation between both parameters suggesting that differences
in the initial slope of CFGE curves (inset of Figure 3) reflect the different number
of dsb induced, as proposed above. In conclusion, the number of dsb induced per Gy
and Da showed a substantial variation for the tumour cells, but not for normal fibroblasts.
As a novelty, GFGE allows to quantify the retention of DNA fragments in the plug (Dahm-Daphi
and Dikomey, 1995). This retention factor, f
ret, depends on cell type, proliferation and presumably other effects. It needs to
be known for calculation of the exact number of dsb. For the tumour cells the retention
factor f
ret ranged between 0.11 (HeLa) and 0.4 (MCF7-Bus) (Table 2), which indicates that
for HeLa cells only 11% of all fragments with a molecular size below 11.5 Mbp were
trapped in the plug in contrast to 40% for MCF7-Bus.
Rejoining of dsbs
Figure 5A
Figure 5
(A) Kinetics of dsb repair in DU145 and LNCaP cells. After irradiation with 60 Gy,
cells were incubated at 37°C for up to 24 h and dsb were measured by CFGE. The FDR
obtained were converted into Gy-equivalents. Data were fitted by nonlinear regression.
(B) Number of dsb 24 h after irradiation with doses up to 150 Gy. For T47D-B8, MCF7-Bus
and HeLa cells, damage measured 24 h after irradiation was higher than the number
of dsb initially induced (dashed lines). Data were fitted by nonlinear regression.
shows the repair kinetics of dsb for DU145 and LNCaP. The FDR as obtained from CFGE
were converted into Gy-equivalents using the induction curves (Figure 3) for calibration.
The repair curves revealed a fast and a slow exponential component with almost identical
half-times for both cell lines (τ
fast=3–4 min and τ
slow=90–100 min). These data illustrate that tumour cells are unlikely to process
dsb with different kinetics compared to normal cells, which was also observed previously
for mouse and rat tumour cells (Dikomey et al, 1995, 1998). However, in LNCaP cells
more dsb were rejoined with fast kinetics than in DU145 and analogously more dsb remain
nonrepaired in DU145 cells.
Residual dsbs
Residual damage was measured in more detail 24 h after irradiation. In order to record
the exact number of dsb, GFGE would be the favorite method; however, the minimum amount
DNA released after 24 h cannot be properly resolved. We thus applied CFGE and came
up with heterogeneous results (Figure 5B). Four tumour cell lines (SCC4451, LNCaP,
MCF7 and MCF7-BB) showed very little residual damage of 8–13 Gy-Eq 24 h after 150 Gy,
while in DU145 and RT112 clearly more dsb remained unrepaired. T47D-B8, MCF7-BUS and
HeLa cells presented values that even exceeded the initial numbers of dsb induced.
The respective gels revealed a DNA smear down to fragment sizes of about 5 kb (not
shown). In those cases, the residual dsb rather reflect apoptotic degradation than
incomplete repair.
Cell cycle distribution
Constant-field gel electrophoresis measurements are known to be dependent on the cell
cycle distribution. S-phase cells show a smaller FDR than G1 cells even for an identical
number of dsb (Dahm-Daphi and Dikomey, 1995). Replication forks are suggested to hinder
DNA from migration. DNA flow cytometry of RT112 cells 24 h after irradiation showed
a cell cycle distribution varying substantially with dose (Figure 6
Figure 6
Cell cycle distribution in RT112 cells 24 h after exposure to doses up to 150 Gy.
Ethanol-fixed cells were PI stained and subjected to DNA flow cytometry.
). In particular, the fraction of S-phase cells increased up to 72% and declined thereafter.
An appreciable variation in cell cycle distribution was also observed for most of
the other strains (Table 1). After 150 Gy, some cell lines showed either more (LNCaP,
MCF-7, MCF7-Bus) or fewer cells in S phase (T47D-B8, MCF7-BB) than the respective
controls. However, even when the cell cycle distribution was the same at 0 and 150 Gy
(DU145, SCC4451), a variation might have occurred at other doses, as observed in RT112.
In conclusion, the measurement of residual damage is variably influenced by the cell
cycle and, thus, do not solely reflect the actual number of dsb.
Correlation between clonogenic radiosensitivity and initial or residual dsbs
Figure 7
Figure 7
Relationship between dsb and cellular radiosensitivity. (A) Number of dsb induced
per Gy and per Da (as calculated from Figure 4) and (B) number of dsb measured 24 h
after 150 Gy (taken from Figure 5B) were plotted vs cellular radiosensitivity expressed
as D
bar (from Table 1).
shows the relationship between the clonogenic radiosensitivity and the number of dsb
induced (A) or 24 h after irradiation with 150 Gy (B). The cell killing (D
bar) was found to be significantly correlated with the number of induced dsb (r
2=0.833, P=0.0015). LNCaP cells were excluded, since the data point was exterior to
the range of 2 s.d. However, the correlation was even significant when LNCaP was included
(r
2=0.47, P=0.041). The data indicate that at least for eight out of nine tumour cells,
the variation in sensitivity could result from differences in the initial damage.
The relationship between residual dsb and sensitivity gave a nonsignificant trend.
This trend, however, was presumably not meaningful, since an increase in the number
of residual dsb was associated with a decrease in sensitivity. In contrast to the
initial damage, residual dsb as determined by CFGE appeared not to be an appropriate
indicator of the clonogenic radiosensitivity.
DISCUSSION
The present study was aimed to define the role of initial and residual dsb on the
radiosensitivity of human tumour cells.
Relationship between induced damage and cellular radiosensitivity
For the nine tumuor cell lines tested, the number of dsb induced was found to vary
by a factor of 2 from 5.75 to 11.0 × 10−12 dsb/Gy/Da (Table 2), which in principle
agreed with most previous studies (Kelland et al, 1988; McMillan et al, 1990; Schwartz
et al, 1990, 1991; Ruiz de Almodovar et al, 1994; Zaffaroni et al, 1994; Whitaker
et al, 1995; Woudstra et al, 1998; Eastham et al, 2001) using either PFGE or neutral
filter elution. Some authors did not mention differences in particular; however, variations
were in the same order of magnitude as the above reports (Giaccia et al, 1992; Olive
et al, 1994; McKay and Kefford, 1995). There was only one report (Olive et al, 1994)
that found differences among six tumour cell lines using neutral filter elution but
factually not by PFGE. Our result is important for three reasons. Firstly, the technique
used is superior to others (see below) and, second, the variation in damage induction
appears to be a property of tumour, but not of normal cells (see below). Most importantly,
for eight out of the nine tumour cell lines the variation in the frequency of induced
dsb showed a significant correlation with the respective variation in radiosensitivity
(Figure 7A). Cell lines with a high number of dsb induced found to be much more sensitive
than cell lines with a low number of induced dsb. This was similarly found by Ruiz
de Almodovar et al (McMillan et al, 1990; Ruiz de Almodovar et al, 1994; Whitaker
et al, 1995), other studies showed an insignificant trend (Schwartz et al, 1988; Giaccia
et al, 1992; Zaffaroni et al, 1994; McKay and Kefford, 1995; Woudstra et al, 1998).
In our study, one (LNCaP, Figure 7A) out of the nine cell lines fell off the general
relationship between initial damage and cell survival indicating that the cellular
radiosensitivity is eventually not only determined by the number of induced dsb but
also by other still unknown factors.
The variations in the dsb induction frequency found for tumour cells are most likely
due to different chromatin structures. High condensation of chromatin structure and
tight DNA–protein association should efficiently protect from oxygen radical attack
to DNA, and vice versa. Such structural variations among tumour cells have in fact
been shown by means of the Halo-assay (Schwartz and Vaughan, 1989; Lynch et al, 1991;
Woudstra et al, 1998). However, direct evidence for chromatin structure being responsible
for differences in the dsb induction among tumour cells is lacking yet. It should
be noted that the large differences in the dsb induction observed are mainly due to
the three extreme values. This may indicate that not all tumour cell lines have an
altered chromatin structure.
Contrary to tumour cells, the seven normal fibroblasts tested showed only scarce variation
in the number of dsb induced, which confirmed previous results of five and twelve
fibroblast lines (Wurm et al, 1994; Dikomey et al, 2000). Taken all together, it can
be assumed that the number of dsb induced varies substantially in tumour cells, but
not in normal human cells. The latter might reflect the general interindividual stability
of the human genome.
Graded vs CFGE
Most measurements of dsb were performed with neutral filter elution, PFGE or CFGE,
which all rely on quantification of overall fragments released from the bulk DNA.
The newly applied GFGE has now the advantage to allow the direct determination of
the number of dsb. It is shown here, for the first time, that this number was in fact
correlated with the initial slope (up to 20 Gy) of the FDR curve of CFGE. However,
this does not mean that the initial slope can reliably taken as an indicator for the
amount of initial damage. In many cases, the variation in the initial slopes of FDR
curves may not be large enough to reveal significant differences between the cell
lines. The initial slope further depend on S-phase cells and on the retention factor
f
ret. High fractions of replicating cells and high retention values decrease the slope
and apparently the number of induced dsb. This might well be the reason why only three
out of 13 studies found a relationship between induced damage and tumour cell radiosensitivity
(Kelland et al, 1988; Schwartz et al, 1988, 1990, 1991; McMillan et al, 1990; Giaccia
et al, 1992; Olive et al, 1994; Ruiz de Almodovar et al, 1994; Zaffaroni et al, 1994;
McKay and Kefford, 1995; Whitaker et al, 1995; Woudstra et al, 1998; Eastham et al,
2001). In conclusion, GFGE is the preferable method over a standard FDR assay to measure
dsb induction in tumour cells.
Number of induced dsb
The mean dsb induction frequency of all 16 cell lines was 8.1 × 10−12 dsb/Gy/Da. (Table
2). This value confirmed our previous results (Dahm-Daphi and Dikomey, 1995; El-Awady
et al, 2001) and also agreed well with other data (range 8–15 × 10−12 dsb/Gy/Da) based
on PFGE, from which a number of dsb can be calculated when it is combined with either
125I-decay, analysis of fragment size distribution after high doses, or restriction
digest (Blöcher and Pohlit, 1982; Ager and Dewey, 1990; Iliakis et al, 1991a, 1991b;
Lawrence et al, 1993; Cedervall et al, 1994, 1995; Löbrich et al, 1994a, 1994b; Rothkamm
and Löbrich, 1999, 2003). Only, Ruiz de Almodovar et al (1994) found a much higher
induction frequency of about 66 dsb × 10−12/Gy/Da.
Residual dsb
The amount of nonrepaired damage showed a broad variation among the nine tumour cell
lines. However, it was shown that apoptosis (Chukhlovin et al, 1995) and also cell
cycle progression could have an impact on dsb measurements, which means that the residual
damage recorded does not exclusively reflect repair capacity. In line with that we
did not find a correlation between residual damage and cell survival, which also agreed
with other reports (Kelland et al, 1988; Olive et al, 1994; McKay and Kefford, 1995;
Nunez et al, 1995; Whitaker et al, 1995; Woudstra et al, 1996). Surprisingly, four
studies found such a correlation but only when the residual damage was measured 1–2 h
after irradiation (Schwartz et al, 1988, 1990; Giaccia et al, 1992; Zaffaroni et al,
1994). It may well be that the impact of cell cycle and apoptosis was minimum after
such short repair intervals and the data, thus, reflect mainly the amount of dsb induced
and initial repair efficiency. Of note, for our data, the association between induced
damage and radiosensitivity appears to be close, which means that the impact of the
repair capacity should be only marginal.
The recently introduced technique to visualise sites of histone γ-H2AX phosphorylation
(Rogakou et al, 1998) needs much lower doses than gel electropheresis to monitor dsb
repair (Rothkamm and Löbrich, 2003) and may thus reduce apoptosis and cell cycle pertubation,
although this technique is generally also sensitive to DNA degradation (Rogakou et
al, 2000) and stalled replication (Ward and Chen, 2001). However, it needs to be shown
whether recording of γ-H2AX will be an advantage over conventional gel electrophoresis
for monitoring residual damage in tumour cells.
In contrast to tumour cells, for normal human and rodent fibroblasts, the variation
in cellular sensitivity did not result from differences in the induction, but the
repair of dsb (Dahm-Daphi et al, 1994; Kiltie et al, 1997; Zhou et al, 1997a, 1997b;
Dikomey et al, 1998, 2000). These results suggest that the mechanisms affecting radiosensitivity
are different for tumour and normal cell lines.
CONCLUSION
Tumour cells vary considerably in their amount of induced damage. These differences
are most likely due to variations of chromatin structure and they may largely account
for tumour cell survival. The number of residual damage was also different among the
cell lines studied, but we could now show that those measurements do not only depend
on the repair capacity per se but also on the cell cycle progression and in some cases
on DNA degradation, presumably due to apoptosis.