Radiotherapy and Oncology 90 (2009) 257–264

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Radiotherapy and Oncology

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Tumor radiosensitivity

The extreme radiosensitivity of the squamous cell carcinoma SKX is due to a

defect in double-strand break repair
Ulla Kasten-Pisula a,1, Apostolos Menegakis b,d,1, Ingo Brammer a, Kerstin Borgmann a, Wael Y. Mansour a,
Sarah Degenhardt a, Mechthild Krause b,d, Andreas Schreiber b,e, Jochen Dahm-Daphi a, Cordula Petersen b,f,
Ekkehard Dikomey a,*, Michael Baumann b,c,d
a
Laboratory of Radiobiology & Experimental Radiooncology, University Medical Center Hamburg – Eppendorf, Hamburg, Germany
b
Dept. of Radiation Oncology, Centre for Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, Technische Universität, Dresden, Germany
c
Experimental Centre, Centre for Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, Technische Universität, Dresden, Germany
d
OncoRay – Centre for Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, Technische Universität, Dresden, Germany
e
Dept. and Practice for Radiotherapy, Städtisches Krankenhaus Dresden-Friedrichstadt, Dresden, Germany
f
Practice for Radiotherapy Hamburg, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Purpose: Squamous cell carcinomas (SCCs) are characterized by moderate radiosensitivity. We have
Received 3 April 2008 established the human head & neck SCC cell line SKX, which shows an exceptionally high radiosensitivity.
Received in revised form 15 October 2008 It was the aim of this study to understand the underlying mechanisms.
Accepted 22 October 2008
Materials & methods: Experiments were performed with SKX and FaDu, the latter taken as a control of
Available online 27 November 2008
moderate radiosensitivity. Cell lines were grown as xenografts as well as cell cultures. For xenografts,
radiosensitivity was determined via local tumour control assay, and for cell cultures using colony assay.
Keywords:
For cell cultures, apoptosis was determined by Annexin V staining and G1-arrest by BrdU labelling. Dou-
Squamous cell carcinoma
Tumour radiosensitivity
ble-strand breaks (DSBs) were detected by both constant-field gel electrophoresis (CFGE) and cH2AX-foci
Double-strand breaks technique; DSB rejoining was also assessed by in vitro rejoining assay; chromosomal damage was deter-
Repair mined by G01-assay.
Prediction Results: Compared to FaDu, SKX cells are extremely radiosensitive as found for both xenografts (TCD50 for
10 fractions 46.0 Gy [95% C.I.: 39; 54 Gy] vs. 18.9 Gy [95% C.I.: 13; 25 Gy]) and cell cultures (D0.01; 7.1 vs.
3.5 Gy). Both cell lines showed neither radiation-induced apoptosis nor radiation-induced permanent G1-
arrest. For DSBs, there was no difference in the induction but for repair with SKX cells showing a higher
level of both, slowly repaired DSBs and residual DSBs. The in vitro DSB repair assay revealed that SKX cells
are defective in nonhomologous endjoining (NHEJ), and that more than 40% of DSBs are rejoined by sin-
gle-strand annealing (SSA). SKX cells also depicted a two-fold higher number of lethal chromosomal aber-
rations when compared to FaDu cells.
Conclusions: The extreme radiosensitivity of the SCC SKX seen both in vivo and in vitro can be ascribed to
a reduced DNA double-strand break repair, resulting from a defect in NHEJ. This defect might be due to
preferred usage of other pathways, such as SSA, which prevents efficient endjoining.
Ó 2008 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 90 (2009) 257–264

For many tumour entities, radiotherapy is known to be a very
potent tool to achieve a local control. This is true especially for
squamous cell carcinomas (SCCs), which are generally character-
ised by moderate radiosensitivity [1,2]. However, even for the
same tumour entity and for an identical treatment protocol, re-
sponse to radiation is often very heterogeneous [3]. Among these
factors defining the tumour response, the cellular radiosensitivity
* Corresponding author. Laboratory of Radiobiology & Experimental Radiooncol-
ogy, Department of Radiotherapy and Radiooncology, University Medical Center
Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany.
E-mail address: dikomey@uke.uni-hamburg.de (E. Dikomey).
1
Shared equal authorship.
as measured in vitro is generally regarded to be one of the most
important parameters [4,5].
The cellular radiosensitivity is principally determined by three
different processes, which are (i) mitosis-linked cell death result-
ing from non- as well as mis-repaired DNA double-strand breaks
(DSBs), which are converted into lethal chromosomal aberrations,
(ii) radiation-induced differentiation implicating a permanent cell
cycle arrest, and (iii) apoptosis [6]. For normal human fibroblasts,
only the first two processes were found to be relevant with an al-
most equal part of both processes [7].
For human tumour cells, the situation appears to be less clear.
There are several reports showing that variation in tumour-cell
radiosensitivity measured in vitro appears to result from differ-

0167-8140/$34.00 Ó 2008 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.radonc.2008.10.019
258 Radiosensitive SCC cell line SKX
ences in both the number of initial and the residual DSBs [8,9].
However, there are also reports suggesting that differences in cel-
lular radiosensitivity result from changes in the level of spontane-
ous or radiation-induced apoptosis [10,11] and/or radiation-
induced differentiation [12]. These inconsistencies indicate that
the contribution of the three processes might not be constant,
but vary from one tumour-cell line to the other [13]. Such a varia-
tion was also seen in vivo. Differences in tumour response observed
after radiotherapy could be associated with the level of proteins
relevant for DSB repair taken as a surrogate of a DSB repair effi-
ciency [14,15], but was also found to correlate with the level of
spontaneous or radiation-induced apoptosis [16–18].
For tumours, there is a great need to understand the mecha-
nisms and processes determining their cellular radiosensitivity in
order to establish a profile that can be used in the clinics to predict
this parameter, and hence the response of the tumour to radiother-
apy. Based on this prediction, an individualisation of therapy might
be possible. This might include the decisions on radiation dose
and/or application of combined treatments.
Information about the mechanisms underlying tumour cell
radiosensitivity is especially obtained, when studying tumours of
extreme high or low sensitivity, because they will give a more
clear-cut picture than the tumours of average sensitivity. This
strategy was shown to result in the important discovery of the
ATM protein and its impact on radiosensitivity as well as the rele-
vance of LigaseIV and DNA-PKcs [19–21].
The present study was initiated to evaluate the extremely high
radiosensitivity of a tumour xenograft, SKX, established from a mod-
erately differentiated squamous cell carcinoma of the floor of mouth
and alveolar bone. Radiosensitivity was assessed by using both
xenografts and cell cultures. We observed that the radiosensitivity
of SKX was due to a substantial DSB repair defect that was based
on a reduced efficiency of the NHEJ pathway and a high frequency
of aberrant repair events. Apoptosis and cell cycle arrest had no
impact.
Materials and methods
Cell lines and culture conditions
Experiments were performed with the squamous cell carcinoma
cell line SKX, which was established from a biopsy taken before
radiotherapy from a moderately differentiated T4, N2, M0 squa-
mous cell carcinoma of the floor of mouth and alveolar bone of
an 83-year old patient at the University Hospital Hamburg-Eppen-
dorf in 1991. The cell line was established by serial transplantation
of tumour pieces in nude mice (NMRI, nu/nu). As the patient died
by intercurrent disease shortly after treatment no follow-up is
available. However, irradiation experiments performed in 1994 at
the University Hospital Hamburg-Eppendorf on SKX tumour xeno-
grafts revealed a high radiosensitivity. The TCD50, i.e. the dose nec-
essary to obtain local tumour control in 50% of the animals, for
single dose irradiation under clamp hypoxia in control nude mice
was 16.6 Gy [95% C.I. 8.6; 24.7 Gy] and in nude mice that were fur-
ther immunosuppressed by WBI with 4 Gy before tumour trans-
plantation was 15.7 Gy [9.8; 21.6] (Baumann and Petersen,
unpublished data). The difference was not statistically significant,
indicating that SKX tumours evoke no or very little immune re-
sponse in nude mice.
To establish the cell line for in vitro experiments, a tumour
xenograft from passage 20 was excised in the Dresden laboratory.
Small tumour pieces were brought into 25 cm2 cell culture flasks
(Nunc) in the incubator. After 30 min, medium was added into
the flask (Dulbecco’s MEM, stable glutamine, 10% FCS, 1 mM pyru-
vate, 1% non-essential amino acids, 20 mM HEPES (Biochrom Ser-
omed), 1% antibiotics (A9909, Sigma). The flasks were incubated
at 37 °C, 5% CO2 and 95% humidity. After 3–4 weeks, cell growth
to a monolayer could be observed. After near confluence, the cells
were further passaged, and cryostocks for further experiments
were established.
For control experiments, we used FaDu cells, which are undif-
ferentiated human hypopharyngeal SCC cells that are characterized
by moderate radiosensitivity [22,23]. For further controls, the hu-
man normal fibroblasts NF47 and the human lymphoblastoid cell
line TK6 were used.
All in vitro experiments except determination of cH2AX foci
were performed in Hamburg, where all cell lines were cultured
in Dulbecco’s modified Eagle’s medium (DMEM) routinely supple-
mented with 10% fetal calf serum and were incubated in a humid-
ified atmosphere of 90% air and 10% CO2 at 37 °C. Evaluation of
cH2AX foci was performed in Dresden, where cells were grown
in Dulbecco’s MEM, stable glutamine, 10% FCS, 1 mM pyruvate,
and 1% non-essential amino acids, 20 mM HEPES, 1% antibiotics.
The flasks were incubated at 37 °C, 5% CO2 and 95% humidity.
Cells in both laboratories were routinely checked for mycoplasma
contamination.
X-irradiation
Cells were irradiated at room temperature or on ice (depending
on the investigated endpoint) with 200 kVp X-rays (Isovolt 320,
Seifert, Ahrensburg, Germany; Hamburg laboratory: 18 mA,
7 mm Be and 0.5 mm Cu filtering; dose rate of 4 Gy/min; Dresden
laboratory:20 mA, 0,5 mm Cu filtering, dose rate 1 Gy/min).
Xenografts and tumour control dose TCD50
Fractionated irradiations were performed using 7–14 weeks old
male and female NMRI (nu/nu) mice from the specific pathogen-
free animal breeding facility of the Experimental Centre of the
Medical Faculty of the University of Dresden. For further immuno-
suppression, animals were whole body irradiated with a dose of
4 Gy (200 kV X-rays, 0.5 mm Cu, 1 Gy/min), 2 days before tumour
transplantation. For the experiments, small tumour pieces were
transplanted into the right hind leg of nude mice (NMRI, nu/nu).
Starting at a diameter of 6 mm, the tumours were locally irradiated
with 10 fractions in 10 days under normal blood flow to total doses
of 5, 10, 20 and 30 Gy (200 kV X-rays, 0.5 mm Cu, 1 Gy/min). The
tumour diameters were measured twice weekly over at least 150
days. The last recurrence was observed at day 104. Recurrences
were scored when the volume increased for at least three consec-
utive measurements after passing a nadir.
Tumour control rates at day 150 after the end of irradiation
were calculated for each dose group after correction for censored
animals according to the method given by Walker and Suit [24]. A
binary (cure/failure) model was used to fit the individual tumour
control data. As in previous investigations reported from this lab-
oratory [25] animals censored later than day 20 after end of treat-
ment were counted as local controls. No animals were censored
before day 20. The results did not change in any meaningful
way when all censored animals were counted as recurrences.
The tumour control probability (TCP) was modeled using the logit
model
TCP ¼ 1=½1 þ expðf ðX;bÞ;
where X represents a vector of covariates that define the treatment,
b a vector of parameters describing radiosensitivity of the tumours,
and f a (possibly nonlinear) function of these. Parameters were esti-
mated using maximum likelihood as implemented in STATA 7.0
software (STATA Corporation, College Station TX). Quoted confi-
dence limits are asymptotic estimates from the results of the likeli-
hood fits. Comparison of maximum likelihood fits was performed
 U. Kasten-Pisula et al. / Radiotherapy and Oncology 90 (2009) 257–264
using the likelihood ratio test [26]. TCD50 at day 150 after the end of
irradiation and associated dose-response curve were determined
from
f ðD; bÞ ¼ b1 ð1  D=b2 Þ;
where ß1 is a constant, and TCD50 = ß2.
Clonogenic assay
Cell survival was measured by colony assay. Cells were trypsin-
ized immediately after the irradiation, and then plated in appropri-
ate numbers using 50 cm2 plastic dishes without feeder cells. After
14 days, cells were fixed, stained with crystal violet, and colonies of
more than 50 cells were scored as ‘survivors’. The surviving frac-
tion of irradiated cells was normalised to the plating efficiency of
non-irradiated controls.
Apoptosis
Apoptosis was detected by vital staining with FITC Annexin
V (BD Pharmingen) and propidium iodide (PI; Calbiochem).
Cells positive only for Annexin V were quantified as early apop-
totic cells, whereas cells with positive staining for both Annex-
in V and PI were classified as late apoptotic or necrotic cells. In
all experiments, cell culture supernatant was collected before
trypsinization. Supernatant and trypsinized cells were combined
before staining, because apoptotic cells detach early from the
surface of culture flasks [27]. Cells were washed twice with
cold PBS (140 mM NaCl, 3 mM KCl, 8 mM Na2HPO4  2 H2O,
1 mM KH2PO4 [pH 7.2]) and resuspended in binding buffer
(10 mM HEPES [pH 7.4], 140 mM NaCl, 2.5 mM CaCl2) at a con-
centration of 1  106 cells/ml. A volume of 100 ll of cell sus-
pension was supplemented with 5 ll of FITC Annexin V and
with 10 ll of a PI stock solution (10 lg/ml). After incubation
for 15 min at RT in the dark, 400 ll of binding buffer was
added. The cells were analyzed by flow cytometry (FACScan,
BD Biosciences).
G1-arrest
The radiation-induced arrest of cells in the G1 phase of the cell
cycle was assessed by continuous BrdU labelling and flow cytome-
try. Exponentially growing cells were irradiated, and immediately
thereafter incubated with 10 lM 5-bromo-20 -deoxyuridine (BrdU;
Serva) to label proliferating cells at replication and with 10 lM 20 -
deoxycytidine (Sigma) to avoid side effects by the BrdU supple-
mentation. Twenty-four hours later, the cells were fixed with
70% ethanol for flow cytometry. At that time, all but arrested cells
had left the G1 phase. The fixed cells were washed with PBS, per-
meabilized with HCl–Triton (2 N HCl, 0.1% Triton-X-100; Serva)
for 30 min at 37 °C, washed twice with 0.1 M sodium borate and
were washed three times with PBS. Thereafter, incubation with
goat serum (Gibco) was carried out for 15 min at RT (1:200 heat-
inactivated goat serum in PBT, 0.5% Tween 20 in PBS). Subse-
quently, cells were incubated with the monoclonal mouse anti-
BrdU antibody (Dako; 10 mg/l in goat serum) for 30 min at RT,
washed twice with PBS, and incubated with the FITC-conjugated
polyclonal secondary goat anti-mouse-immunoglobulin antibody
(Dako; 25 lg/l in goat serum) for 30 min at RT. After two washing
steps with PBS, cells were counterstained with 15 lM propidium
iodide (PI) for at least 20 min at RT. The stained cells were analysed
by flow cytometry (FACScan, BD Biosciences) measuring the fluo-
rescence of both anti-BrdU-FITC and PI. From the resulting dot
plots, the fraction of BrdU-unlabelled (i.e. arrested) G1 cells was
derived by using the CellQuest Pro computer program (BD
Biosciences).
259
Double-strand breaks
Double-strand breaks (DSBs) were detected by either constant-
field gel electrophoresis (CFGE) or cH2AX-foci technique using
nearly confluent cell cultures. CFGE was carried out according to
Dahm-Daphi and Dikomey [28]. Briefly, nearly confluent cells were
irradiated on ice either in agarose plugs for determination of DSB
induction, or as monolayer for determination of DSB repair (follow-
ing repair incubation at 37 °C). After plug preparation, cells were
lysed (0.4 M EDTA, 2% Na-N-laurylsarcosine, 1 mg/ml proteinase
K [pH 8]; 30 min on ice, thereafter 16 h at 37 °C). After washing
three times with TE-buffer (10 mM Tris–HCl, 1 mM EDTA [pH 8]),
plugs were inserted into an agarose gel. Subsequently, electropho-
resis was started (40 h, RT, 0.6 V/cm, 0.5 TBE-buffer, 45 mM Tris–
borate, 1 mM EDTA [pH 8]). DNA was stained with ethidium bro-
mide (0.5 lg/ml) and destained in water. For the quantitative eval-
uation of the DNA distribution, ethidium bromide was excited with
302 nm light, and simultaneously a video picture was taken using a
CCD camera (Sony XC-75CE). This picture was analysed densito-
metrically (OPTIMAS Corporation, Seattle, USA) to quantify the
amount of DNA. The fraction of DNA released from the plug into
the gel (Frel) was calculated by Frel = frel/(fplug + frel), where
fplug and frel corresponded to the relative fluorescence of the
DNA remaining in or released from the plug, respectively.
For detection of DSBs via cH2AX-foci, confluent cell cultures
were irradiated, trypsinised after different time intervals and
placed on glass slides by cytospin procedure (75,000 cells per spot;
200 rpm, 2 min), and then fixed in neutral buffered 4% formalin for
15 min. After permeabilization of cell membranes with Triton X-
100 (0.01% in PBS v/v; three times), bovine serum albumin (1% in
PBS, RT, 30 min) was used to block unspecific reactions before pri-
mary antibody was added for 1 h (Upstate; clone JBW301; 1:1000
in 1% BSA/PBS; RT). As secondary antibody ALEXA 594 fluorescent
probe was used (Molecular Probes; 1:400; RT, 30 min). Nuclei were
labelled by DAPI (Boehringer; 25 lg/ml; RT, 10 min). The cH2AX-
foci were counted visually using a Zeiss fluorescence microscope
(Zeiss Axioplan 2) with red fluorescence signal (filter set: 15, exci-
tation 546 nm, emission: 590 nm) under 630-fold magnification.
The cells were selected according to morphological criteria, and
only intact nuclei (checked via DAPI staining using filter set: 2,
excitation: 365 nm, emission: 420 nm) were analysed. For evalua-
tion, 200 nuclei of each dose group were randomly chosen, and the
number of foci per nucleus was recorded.
In vitro DSB endjoining
For in vitro end joining experiments, whole-cell extracts were
prepared as described [32]. 108 cells of each cell line were har-
vested, washed twice with ice-cold PBS and were lysed on ice in
4 packed cell volume of hypotonic buffer (10 mM Tris–HCl pH
8.0; 1 mM EDTA; 5 mM DTT; Protease inhibitors: 100 mM PMSF;
1 mM leupeptin; 1 mM pepstatin A) until only nuclei are visible
(90%). Cells were then lysed by adding about 2 ml of buffer
(50 mM Tris–HCl, 10 mM MgCl2, 2 mM DTT, 25% sucrose, 50% glyc-
erol, pH7.0) and 400 ll of 3.9 M ammonium sulphate and stirred
slowly for 30 min at 4 °C. The lysates were cleared from insoluble
material and genomic DNA by ultracentrifugation at 40,000 rpm
for 1 h at 2 °C. After ammonium sulphate precipitation, the extracts
were dialysed against dialysis buffer (30 mM Tris–HCl pH 8.0;
90 mM KCl; 10 mM b-Na-glycerophosphate pH 7.0; 2 mM EGTA
pH 8.5; 1 mM EDTA pH 8.0; 1 mM DTT; 2 mM MgCl2; 20% Glycerol;
protease inhibitors (1 PMSF/leupeptin/pepstatin)) for 3 h at 4 °C.
Extracts typically yield a protein concentration of 6–8 lg/ll and
were aliquoted and stored in liquid nitrogen for further use. Di-
rectly before the endjoining reactions, the required extract volume
was dialysed on micro dialysis filters (0.025 lm; Millipore,
260
Germany) for 30 min at 4 °C against freshly prepared MOPSO buf-
fer (50 mM MOPSO–NaOH pH 7.5; 40 mM KCl; 10 mM MgCl2;
5 mM 2-mercaptoethanol).
For endjoining reactions, the plasmid substrate pEJSSA was
cleaved by HindIII/PstI and gel purified. The double-digest creates
non-compatible ends with 30 and 50 overhangs and gel purified.
Forty-five ng of linear plasmid was incubated for 6 h at 25 °C in a
total volume of 10 ll containing 50 lg of extracted protein in reac-
tion buffer supplemented with 1 mM ATP pH 7.5; 200 lM dNTPs
(50 lM each) and 50 ng/ll BSA. Reactions were terminated by
adjustment to 20 mM Tris–HCl pH 7.5, 10 mM EDTA, 1% sodium
dodecyl sulfate (SDS) and incubation at 65 °C for 10 min. DNA
was then purified (Blood and Tissue kit, Qiagen), and 10 ll were
transformed into E.coli (DH5a EC100, Epicentre Biotechnologies)
by electroporation. Bacteria were plated on LB agar plates with
kanamycin, and incubated at 37 °C overnight to yield single clones.
In order to further characterize the repair results, a PCR was carried
out directly from individual clones using primers P1 and P2, and
analysed on agarose gels as described previously [33].
Chromosomal aberrations
Chromosomal damage was assessed by G01-assay as described
previously [7]. Cells grown to near confluence were irradiated at
RT, followed by a delayed plating 14 h after irradiation. After
growth for 44 h, colcemid (0.2 lg/ml; Gibco) was added for 8 h.
Thereafter, cells were treated with hypotonic KCl solution
(0.075 M; Sigma), and were fixed several times in Carnoy’s fixative.
Fixed cells were dropped on precleaned wet slides, stained with 2%
Giemsa (Sigma) for 7 min and embedded permanently with Ente-
llan (Merck).
Analysis of metaphase fragments was performed by means of
light microscopy. Routinely, three to four encoded slides with
25–50 cells per slide were scored using a Zeiss microscope. Meta-
phase spreads were screened for excess fragments arising from ter-
minal or interstitial deletions and incomplete changes as well as
dicentric chromosomes, whereby each dicentric was considered
to be associated with one acentric fragment.
Data evaluation
Experiments were repeated up to five times and data were gi-
ven as a mean (±SEM). For analysing and graphing the data, the
Prism software (GraphPad Prism) was used. For plasmid rejoining,
A 60
50
40
TCD50 (Gy)
30
20
10
0 10 -3
SKX FaDu
Fig. 1. Radiosensitivity of SKX and FaDu cells grown either as (A) xenografts or (B) cell cultures. (A) Tumor control dose 50% (TCD50) for SKX tumours obtained for irradiation
with 10 fractions within 10 days under ambient blood flow compared to the previously published data for the same treatment in FaDu tumours [29]. Error bars show 95% C.I.
(B) Cell survival determined by colony assay. Data were fitted by non-linear regression. Error bars represent standard error of the mean.
Radiosensitive SCC cell line SKX
difference in the efficiency was tested by Mann–Witney test, and
for pathways distribution by Fishers exact test.
Results
Radiosensitivity
Fig. 1 demonstrates the very high radiosensitivity of SKX cells
growing either (A) as xenografts or (B) as cell cultures. For xeno-
grafts, the tumour control dose 50% (TCD50) was determined after
10 fractions in 10 days, and was compared to respective data pre-
viously published for FaDu [29]. SKX showed a very high radiosen-
sitivity with a TCD50 value of 19.8 Gy (95% C.I. 13; 25) in contrast to
the intermediate radiosensitivity of FaDu with a TCD50 of 46 Gy
(39; 54).
For cell cultures, cells were grown to nearly plateau phase, irra-
diated at RT, and immediately plated for colony assay. The data
were analysed by the linear quadratic equation S = exp(aDbD2)
in order to determine the dose required to reduce the survival to
1%, D0.01. For SKX cells, this value was 3.5 Gy in contrast to
7.1 Gy for FaDu cells. A similar difference was seen after delayed
plating. These data clearly demonstrate that under both conditions,
xenograft and cell culture, SKX cells are twofold more sensitive
than FaDu cells.
Apoptosis and G1-arrest
In order to elucidate the reason for the different sensitivity, we
determined the incidence of apoptosis using Annexin V staining
and flow cytometry. For non-irradiated cultures, the fraction of
apoptotic cells was higher in SKX than in FaDu cells, amounting
to 21 compared to 3% (Fig. 2). However, when irradiated with
6 Gy, for both cell lines there was no further increase in this frac-
tion neither at 12 h nor at 24 h after irradiation (Fig. 2). In contrast,
for the lymphoblastoid cell line TK6, which was used as a positive
control, there was a clear-cut increase of the percentage of apopto-
tic cells from 26% to 47% when irradiated with 6 Gy.
We next determined by BrdU labelling and flow cytometry
whether differences in cell cycle progression exist which possibly
cause the distinct radiosensitivities. Without irradiation, the SKX
strain but not FaDu showed a substantial number of cells arrested
in G1 (Fig. 3). For both strains, these fractions did not increase after
irradiation with 1 or 6 Gy. In contrast, normal human fibroblasts
B 1
10 -1
Survival
10 -2
FaDu
SKX
0 1 2 3 4 5 6 7 8 9
X-ray dose (Gy)
 U. Kasten-Pisula et al. / Radiotherapy and Oncology 90 (2009) 257–264
60
non-irradiated
50 6 Gy → 12 h
Fraction of apoptotic cells,%
6 Gy → 24 h
40
30
20
10
0 SKX is twofold higher when compared to FaDu cells.
SKX FaDu
Fig. 2. Radiation-induced apoptosis in SKX and FaDu cells. Cells were irradiated
with 6 Gy of X-rays followed by an incubation at 37 °C for 12 or 24 h. Apoptosis was
detected by Annexin V staining and flow cytometry. Lymphoblastoid cell line TK6
was used as a positive control. Error bars represent standard error of the mean.
NF47 were significantly arrested in G1 upon irradiation. These data
demonstrate that both cell lines, SKX and FaDu, do execute neither
radiation-induced apoptosis nor a G1-arrest, which means that the
pronounced difference in cellular radiosensitivity cannot be ex-
plained by either of these mechanisms.
Double-strand break repair
Fig. 4 presents the data obtained for both the induction and repair
of DNA double-strand breaks (DSBs), as detected either by CFGE or
cH2AX-foci technique. For both cell lines, the fraction of DNA re-
leased as measured with CFGE increases with increasing dose, with
no difference between the two cell lines, indicating that the number
of DSBs induced per Gy is the same for SKX and FaDu cells (Fig. 4A).
There is, however, a clear difference in the repair kinetics as deter-
mined by CFGE after irradiation with 40 Gy (Fig. 4B). The fraction
of DNA released as measured for different repair intervals was con-
verted into a relative number of DSBs using the data of Fig. 4A for cal-
ibration. For both cell lines, the number of DSBs declines with
biphasic kinetics down to the level of residual DSBs, which is about
4% for FaDu but even 15% for SKX cells. Further analysis of the kinet-
ics revealed that for FaDu cells, 69% of all DSBs are repaired with fast
Fraction of cells arrested in G1 phase, %
0 Gy → 24 h
30 1 Gy → 24 h
6 Gy → 24 h
20
10
0 tion after exposure to X-irradiation.
SKX FaDu
Fig. 3. Radiation-induced G1-arrest in SKX and FaDu cells. Cells were irradiated
with 1 or 6 Gy of X-rays followed by continuous incubation with BrdU for 24 h.
Fraction of cells arrested in G1 (without BrdU-incorporation) was determined by
flow cytometry. Normal fibroblast cell line NF47 was used as a positive control.
Error bars represent standard error of the mean.
261
kinetics with a half-time of about 5 min, and 27% with slow kinetics
with a half-time of 130 min. For SKX cells, the respective fractions
are 44 and 41%, with half-times of 15 and 180 min, respectively.
These data demonstrate that SKX cells have a substantial DSB repair
defect leading to prolonged repair half-times, elevated fractions of
slowly rejoined and, most importantly, of residual DSBs. Similar
observations were made, when DSBs were detected by cH2AX-foci
technique (Fig. 4C). For this experiment cells were irradiated with
4 Gy, and the first time point chosen was 30 min after irradiation.
For SKX cells, there is again a pronounced increase in the number
of both slowly rejoined as well as residual cH2AX-foci.
Fig. 4D shows the difference in residual DSBs as detected via
cH2AX-foci as a function of dose. For FaDu cells, there is only a
moderate increase with dose, while there is a steep increase for
SKX cells. On an average, the number of residual DSBs present in
TK6 Substantially reduced DSB repair capacity is likely due to a de-
fect in non-homologous endjoining (NHEJ) [30,31]. In order to fur-
ther characterize the DSB repair defect, whole cell extracts (WCE) of
both cell lines were prepared and used for in vitro endjoining [32] of
the linearized plasmid pEJSSA [33]. After repair incubation the plas-
mid DNA was transformed into E. coli. Only repair that has led to cir-
cular products gives rise to bacterial colonies (Fig. 5A). SKX cell
extracts showed a more than 9-fold reduced capability to form
these circles as compared to FaDu (Fig. 5B). The pEJSSA can be re-
circularized via NHEJ or homology-dependent single-strand
annealing (SSA) [33]. In order to determine which pathway was
used, the repair region was amplified by PCR for 84 individual col-
onies. Repair results of NHEJ lead to a PCR fragment of about 570 bp
and SSA to exactly 415 bp (Fig. 5C). FaDu extracts used NHEJ for 88%
and SSA for 12% of repair junctions. In contrast, SKX employed NHEJ
for only 57%, and the more mutagenic SSA pathway for 43% of repair
events (Fig. 5D). Together these results corroborate the general DSB
repair defect of SKX cells as described before and confirm that
mainly the endjoining pathway is affected. In addition, the balance
between both pathways is shown to be significantly shifted in SKX
cells towards usage of SSA when compared to FaDu.
Chromosomal damage
Fig. 6A shows the difference in chromosomal damage between
SKX and FaDu cells as determined via G01-technique (Fig. 6A).
Again, there is only a moderate increase of chromosomal aberra-
tions with dose for FaDu cells, but a steep rise for SKX cells. When
survival as taken from Fig. 1B is plotted against the respective
number of these aberrations (Fig. 6A), it can be seen that the asso-
ciation between these two parameters is the same for both cell
lines and can be described by one straight exponential decline
(Fig. 6B; r2 = 0.99). It can also be taken from Fig. 6B that for both
cell lines the induction of one chromosomal aberration leads to a
reduction in survival to about 37% (dotted lines). This finding dem-
onstrates – assuming that the chromosomal aberrations are dis-
tributed according to a Poisson equation – that almost all cell
inactivation observed after X-irradiation has to be ascribed to these
aberrations, and that the difference in radiosensitivity between
these two cell lines can solely be explained by the higher number
of aberrations formed in SKX cells. This result is in line with the
observations made above that for both, SKX and FaDu cells, neither
apoptosis nor G1-arrest played a substantial role for cell inactiva-
NF47
Discussion
Human head and neck squamous cell carcinoma experiences
usually a moderate clinical radiosensitivity. As an exception, the
262 Radiosensitive SCC cell line SKX

1.0
40 Gy → 37°C

Number of double-strand breaks (%)

100

Fraction of DNA released 0.8 SKX 60 SKX

FaDu FaDu
30
0.6

10
0.4
6

0.2 3

A B
0.0 1
0 20 40 60 80 0 6 12 18 24
X-ray dose, Gy Time at 37°C after irradiation, h

30
100 4 Gy → 37°C X → 37°C, 24h
Number of γH2AX- foci per cell

Number of γH2AX-foci per cell

25
SKX SKX
FaDu FaDu
20

15
10

10

5
C D
1 0
0 6 12 18 24 0 2 4 6 8
Time at 37°C after irradiation, h X-ray dose (Gy)

Fig. 4. Induction and repair of DNA DSBs in SKX and FaDu cells. DSBs were detected either by (A and B) CFGE or (C and D) cH2AX-foci technique. (A) Induction of DSBs as
determined immediately after irradiation on ice using CFGE; (B and C) Repair kinetics of DSBs measured either after irradiation with 40 Gy using CFGE or with 4 Gy using the
cH2AX-foci technique; (D) Residual DSBs as analysed by the cH2AX-foci technique 24 h after irradiation. Data were fitted by non-linear regression. Error bars represent
standard error of the mean.

HNSCC cell line SKX is characterized by a remarkably high radio-
sensitivity when grown as xenograft in nude mice or as cell cul-
ture. The aim of this study was to understand the mechanisms
underlying this high radiosensitivity.
In a previous study with 9 different tumour cell lines, the num-
ber of DSBs induced per Gy was demonstrated to vary substan-
tially, which was considered to be the major reason for the
respective differences in cellular radiosensitivity observed [8].
However, for SKX cells the extreme radiosensitivity cannot be as-
cribed to a higher number of DSBs induced, since there was no dif-
ference between this cell line and FaDu cells, which were used as a
control (Fig. 4A).
In contrast, analysis of the repair kinetics revealed, that SKX
cells have a clear defect in DSB repair leading to both, an increased
fraction of slowly repaired and residual DSBs (Fig. 4B and C). At
40 Gy (Fig. 4B), these numbers of residual DSBs are equivalent to
a DSB repair capacity of 95% for FaDu cells and 85% for SKX cells.
Biphasic kinetics as seen here for DSB repair in SKX and FaDu cells
were also reported for other cell lines [30,34]. Generally, 30% of all
DSBs are rejoined with slow kinetics as also found here for FaDu
cells [30,34]. In SKX cells, this fraction amounted to 41%. Such an
elevated fraction was also seen for other cell lines with a known
DSB repair defect [35].
The pronounced differences in the DSB repair kinetics observed
for SKX cells suggest that this cell line is defective in non-homolo-
gous endjoining (NHEJ), which is the major DSB repair pathway ac-
tive in mammalian cells [36]. The specific defect in NHEJ, however,
is not known yet, and is the subject of an ongoing project.
The DSB repair defect was found to translate into an elevated
number of lethal chromosomal aberrations (Fig. 6A). The relation-
ship between chromosomal damage and cell survival was found to
be the same for both cell lines (Fig. 6B). This result illustrates that
the increase in chromosomal aberrations measured for SKX cells
completely matches with the respective decrease in cell survival.
In order to further characterize the repair defect, we chose an
in vitro endjoining assay using thoroughly extracted tumour cell
proteins [32]. In vitro endjoining of DSB in plasmid DNA generally
lead to linear dimers and multimers but also to circular plasmid
DNA, which is well established to particularly reflect pro- or defi-
ciency in ‘‘classical” DNA-PK-dependent NHEJ [32]. The DNA sub-
strate applied allowed us further to monitor the usage of SSA
instead of NHEJ for DSB repair. We found that the efficiency of
‘‘classical” NHEJ was drastically reduced in SKX cells. This differ-
ence between both cell lines in vitro was even larger than the de-
fect found in intact cells. This can be explained at least by the
shift of the SKX cells to repair more than 40% DSB via SSA. For hu-
 U. Kasten-Pisula et al. / Radiotherapy and Oncology 90 (2009) 257–264 263

A C SKX FaDu
cell free 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
extracts
repair bacterial
incubation colonies
linear
pEJSSA

B 600 D 100
SSA
Number of bacterial colonies

500

Percentage of pathways, %
80 SSA
p=0.017
400
60
300
p=0.016 NHEJ
40
200
NHEJ
20
100

0 0
SKX FaDu SKX FaDu

Fig. 5. In vitro endjoining by extracts of SKX and FaDu cells. (A) HindIII/Pst linearized plasmids pEJSSA were incubated for 2 h with whole cell protein extracts of either strain.
The DNA was then transformed into E coli and colonies were counted, individually harvested and subjected to PCR amplification. (B) Efficiency of plasmid joining to form
circular products as measured by the number of bacterial colonies. (C) Example of PCR products of individual colonies. Fragment sizes of about 570 bp correspond to NHEJ
which are the dominant events in FaDu. SSA lead to exact fragment size of 415 bp (lanes 3, 5, 7, 10, 12) (lane 1, positive control of uncleaved plasmid). (D) Percentage of
linearised plasmid pEJSSA rejoined either by NHEJ or SSA.

A B
12 1
Chromosome aberrations per cell

10

8 10 -1
SKX SKX
Survival

FaDu FaDu
6

4 10 -2

2
r²=0.99
p<0.0001
0
10 -3
0 2 4 6 0 2 4 6 8
X-ray dose (Gy) Chromosome aberrations per cell

Fig. 6. (A) Lethal chromosomal aberrations in X-irradiated SKX and FaDu cells. Chromosomal damage was determined by the G01-assay. (B) Relationship between
chromosomal damage (data from Fig. 5A) and cell survival (data from Fig. 1B) in SKX and FaDu cells. Data were fitted by non-linear (A) or linear (B) regression. Error bars
represent standard error of the mean.

man tumour cells, such a drastic shift towards SSA is described joining. The first explanation appears unlikely since all ‘‘core” end-
here for the first time. By definition, SSA is a mutagenic pathway, joining components of SKX cells showed normal expression and
which involves deletion of long stretches of DNA flanking the activity (data to be published elsewhere). However, further studies
DSB [36]. Various degrees of error-prone in vitro endjoining were are needed to elucidate whether for instance high exonucleolytic
also observed for other tumour cells. In human bladder cancer activity compromises the stability of DNA ends in SKX cells which
cells, DNA double-strand break repair was found to be error prone may likewise result in deficient endjoining and strand resection as
and to involve microhomology-associated endjoining [37]. An im- initial step towards SSA [40]. It can be speculated that after irradi-
paired NHEJ was also seen in mismatch repair-deficient colon car- ation this end degradation precludes rejoining of the ends, if suit-
cinomas [38] and also in human head and neck cancer [39], but up able homologous repeat sequences are not available.
to now no preferred use of SSA is reported. It is possible that either Upon irradiation both cell lines, SKX and FaDu, showed neither
the failure to properly perform NHEJ guides the repair towards SSA an increase in apoptosis nor a permanent G1-arrest (Figs. 2 and 3).
or conversely that initiation of SSA may prevent successful end- This is in line with the fact that these cell lines are either deficient
264 Radiosensitive SCC cell line SKX
in p53 as shown for FaDu [41] or mutated as in SKX cells (data not
shown). It was also noted that non-irradiated SKX cells already de-
pict a substantial fraction of both apoptotic cells and cells arrested
in the G1 phase (Figs. 2 and 3) and also a higher incidence of foci in
untreated cells (Fig. 4D) which was not seen for non-irradiated
FaDu cells. Probably, these effects also result from the DSB repair
defect as described above. DSBs are also produced in non-irradi-
ated cells either during transcription or replication. In SKX cells,
some of these DSBs might not be repaired or misrepaired and will
then give rise to apoptosis and/or G1-arrest. This concept, however,
implies that the spontaneous apoptosis and G1-arrest as seen in
SKX cells do not require wtp53, as already suggested in other re-
ports [17,42–45]. Together, these data indicate that the loss of pro-
liferative capacity of both tumor cell lines after X-irradiation solely
results from mitotic cell death, while neither G1-arrest nor apopto-
sis has a significant impact.
In conclusion, it was shown here that the remarkable radiosen-
sitivity of the SKX cell line can be exclusively attributed to a defec-
tive DSB repair resulting in an enhanced formation of lethal
chromosomal aberrations, while other processes such as apoptosis
or permanent G1-arrest are not relevant. The deficient DSB repair
in SKX cells is due to a defect in NHEJ coupled with a shift towards
usage of SSA.
Acknowledgements
The authors greatly acknowledge the technical assistance of B.
Riepen, C. Hausmann and I. Schrauth for their technical assistance.
References
[1] Halperin EC, Perez CA, Brady LW. Perez and Brady’s principles and practice of
radiation oncology. New York: Lippincott Williams & Wilkins; 2007.
[2] Lohr F, Wenz F. Strahlentherapie kompakt. München: Urban & Fischer; 2003.
[3] Peters LJ, Withers HR, Thames Jr HD, Fletcher GH. Tumor radioresistance in
clinical radiotherapy. Int J Radiat Oncol Biol Phys 1982;8:101–8.
[4] Deacon J, Peckham MJ, Steel GG. The radio responsiveness of human tumours
and the initial slope of the cell survival curve. Radiother Oncol 1984;2:317–23.
[5] West CML, Davidson SE, Roberts SA, Hunter RD. The independence of intrinsic
radiosensitivity as a prognostic factor for patient response to radiotherapy of
carcinoma of the cervix. Br J Cancer 1997;76:1184–90.
[6] Dikomey E, Borgmann K, Brammer I, Kasten-Pisula U. Molecular mechanisms
of individual radiosensitivity studied in normal diploid human fibroblasts.
Toxicology 2003;193:125–35.
[7] Borgmann K, Dede M, Wrona A, Brammer I, Overgaard J, Dikomey E. For X-
irradiated normal human fibroblasts only half of cell inactivation results from
chromosomal damage. Int J Radiat Oncol Biol Phys 2004;58:445–52.
[8] El-Awady RA, Dikomey E, Dahm-Daphi J. Radiosensitivity of human tumour
cells is associated with the induction but not with the repair of DNA double-
strand breaks. Br J Cancer 2003;89:593–601.
[9] Akudugu JM, Theron T, Serafin AM, Böhm L. Influence of DNA double-strand
break rejoining on clonogenic survival and micronucleus yield in human cell
lines. Int J Radiat Biol 2004;80:93–104.
[10] Pruschy M, Rocha S, Zaugg K, et al. Key targets for the execution of radiation-
induced tumor cell apoptosis: the role of p53 and CASPASES. Int J Radiat Oncol
Biol Phys 2001;49:561–7.
[11] Ferlini C, D’Amelio R, Scambia G. Apoptosis induced by ionizing radiation. The
biological basis of radiosensitivity. Sub Cell Biochem 2002;36:171–86.
[12] Bromfield GP, Meng A, Warde P, Bristow RG. Cell death in irradiated prostate
epithelial cells: role of apoptotic and clonogenic cell kill. Prostate Cancer
Prostatic Dis 2003;6:73–85.
[13] Abend M, Rhein A, Gilbertz KP, Blakely WF, vanBeuningen D. Correlation of
micronucleus and apoptosis assays with reproductive cell death. Int J Radiat
Biol 1995:315–26.
[14] Komuro Y, Watanabe T, Hosoi Y, et al. The expression pattern of Ku correlates
with tumor radiosensitivity and disease free survival in patients with rectal
carcinoma. Cancer 2002;95:1199–205.
[15] Harima Y, Sawada S, Miyazaki Y, et al. Expression of Ku80 in cervical cancer
correlates with response to radiotherapy and survival. Am J Clin Oncol
2003;26:e80–5.
[16] Rödel C, Grabenbauer GG, Rödel F, et al. Apoptosis p53, BCL-2, and KI-67 in
invasive bladder carcinoma: possible predictors for respoonse to
radiochemotherapy and successful bladder preservation. Int J Radiat Oncol
Biol Phys 2000;46:1213–21.
[17] Rödel C, Grabenbauer GG, Papadopoulos T, et al. Int J Radiat Oncol Biol Phys
2002;52:294–303.
[18] Bandoh N, Hayashi T, Kishibe K, et al. Prognostic value of p53 mutations, bax,
and spontaneous apoptosis in maxillary sinus squamous cell carcinoma.
Cancer 2002;94:1968–80.
[19] Arlett CF. Human cellular radiosensitivity – the search for the diagnostic holy
grail or a poisoned chalice. Adv Radiat Biol 1992;16:273–92.
[20] Allalunis-Turner MJ, Zia PKY, Barron GM, Mirzayans R, Day RS. Radiation-
induced DNA damage and repair in cells of a radiosensitive human malignant
glioma cell line. Radiat Res 1995;144:288–93.
[21] Badie C, Goodhardt M, Waugh A, et al. A DNA double-strand break defective
fibroblast cell line (180BR) derived from a radiosensitive patient represents a
new mutant phenotype. Cancer Res 1997;57:4600–7.
[22] Suit HD, Zietman A, Tomkinson K, Ramsay J, Gerweck L, Sedlacek R. Radiation
response of xenografts of a human squamous cell carcinoma and a
glioblastoma multiforme: a progress report. Int J Radiat Oncol Biol Phys
1990;18:365–73.
[23] Toulany M, Dittmann K, Krüger M, Baumann M, Rodemann HP.
Radioresistance of K-Ras mutated human tumor cells is mediated through
EGFR-dependent activation of PI3K-AKT pathway. Radiother Oncol
2005;76:141–50.
[24] Walker AM, Suit HD. Assessment of local tumor control using censored tumor
response data. Int J Radiat Oncol Biol Phys 1983;9:383–6.
[25] Baumann M, Krause M. Targeting the epidermal growth factor receptor in
radiotherapy: radiobiological mechanisms, preclinical and clinical results.
Radiother Oncol 2004;72:257–66.
[26] Edwards AWF. Likelihood. Baltimore: John Hopkins University Press; 1992.
[27] Darzynkiewicz Z, Juan G, Li X, Gorczyca W, Murakami T, Traganos F. Cytometry
in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis).
Cytometry 1997;27:1–20.
[28] Dahm-Daphi J, Dikomey E. Rejoining of DNA double-strand breaks in X-
irradiated CHO cells studied by constant- and graded-field gel electrophoresis.
Inter J Radiat Biol 1996;69:615–21.
[29] Appold S, Baumann M, Petersen C, Horn K, Eichhorn F. Comparison of the
response of human FaDu squamous cell carcinoma in nude mice after
hypofractionated-accelerated regimens and ‘‘curative” fractionation
schedules. Strahlenther Onkol 1998;174:315–9.
[30] Kasten-Pisula U, Tastan H, Dikomey E. Huge differences in cellular
radiosensitivity are due to only very small variations in double-strand break
repair capacity. Inter J Radiat Biol 2005;81:409–19.
[31] Lieber MR, Ma Y, Pannicke U, Schwarz K. Mechanism and regulation of human
non-homologous DNA end-joining. Nat Rev 2003;4:712–20.
[32] Kuhfittig-Kulle S, Feldmann E, Odersky A, et al. The mutagenic potential of
non-homologous end joining in the absence of the NHEJ core factors Ku70/80,
DNA-PKcs and XRCC4-LigIV. Mutagenesis 2007;22:217–33.
[33] Mansour WY, Schumacher S, Rosskopf R, et al. Hierarchy of nonhomologous
end-joining, single-strand annealing and gene conversion at site-directed DNA
double-strand breaks. Nucleic Acids Res 2008;36:4088–98.
[34] Wang H, Zeng ZC, Bui TA, et al. Efficient rejoining of radiation-induced DNA
double-strand breaks in vertebrate cells deficient in genes of the RAD52
epistasis group. Oncogene 2001;20:2212–24.
[35] Kasten U, Borgmann K, Burgmann P, Li G, Dikomey E. Overexpression of
human Ku70/Ku80 in rat cells resulting in reduced dsb repair capacity with
appropriate increase in cell radiosensitivity but with no effect on cell recovery.
Radiat Res 1999;151:532–9.
[36] Pfeiffer P, Goedecke W, Obe G. Mechanisms of DNA double-strand break repair
and their potential to induce chromosomal aberrations. Mutagenesis
2000;15:289–302.
[37] Bentley J, Diggle CP, Harnden P, Knowles MA, Kiltie AE. DNA double strand
break repair in human bladder cancer is error prone and involves
microhomology-associated end-joining. Nucleic Acids Res 2004;32:
5249–59.
[38] Koh KH, Kang HJ, Li LS, et al. Impaired nonhomologous end-joining in
mismatch repair-deficient colon carcinomas. Lab Invest 2005;85:1130–8.
[39] Shin KH, Kang MK, Kim RH, Kameta A, Baluda MA, Park NH. Abnormal DNA
end-joining activity in human head and neck cancer. Inter J Mol Med
2006;17:917–24.
[40] Frank-Vaillant M, Marcand S. Transient stability of DNA ends allows
nonhomologous end joining to precede homologous recombination. Mol cell
2002;10:1189–99.
[41] Eicheler W, Zips D, Dorfler A, Grenman R, Baumann M. Splicing mutations in
TP53 in human squamous cell carcinoma lines influence
immunohistochemical detection. J Histochem Cytochem 2002;50:197–204.
[42] Shi YQ, Wuergler FE, Blattmann H, Crompton NE. Distinct apoptotic
phenotypes induced by radiation and ceramide in both p53-wild-type and
p53-mutated lymphoblastoid cells. Radiat Environ Biophys 2001;40:
301–8.
[43] Jesionek-Kupnicka D, Tenderenda M, Rutkowski P. Extent of spontaneous
apoptosis in gastric cancer: relation to proliferative index, p53 expression, CD
34 expression and histopathological features. J Exp Clin Cancer Res
2002;21:371–5.
[44] Tenjo T, Toyoda M, Okuda J, et al. Prognostic significance of p27(kip1) protein
expression and spontaneous apoptosis in patients with colorectal
adenocarcinomas. Oncology 2000;58:45–51.
[45] Zschenker O, Borgmann K, Streichert T, Meier I, Wrona A, Dikomey E.
Lymphoblastoid cell lines differing in p53 status show clear differences in
basal gene expression with minor changes after irradiation. Radiother Oncol
2006;80:236–49.