Int. J. Radiat. Biol., Vol. 81, No. 2, February 2005, pp. 125 – 138
ORIGINAL ARTICLE
Dose – response for radiation-induced apoptosis, residual 53BP1 foci
and DNA-loop relaxation in human lymphocytes
J. TORUDD1, M. PROTOPOPOVA2, R. SARIMOV1,3, J. NYGREN4,5, S. ERIKSSON5,
E. MARKOVÁ1,6, M. CHOVANEC1,6, G. SELIVANOVA2 and I. Y. BELYAEV1,3
1
Department of Genetics, Microbiology and Toxicology, Stockholm University, Stockholm, Sweden 2Cancer Centrum
Karolinska, Karolinska Institutet, Stockholm, Sweden 3Laboratory of Radiobiology, General Physics Institute, Russian
Academy of Science, Moscow, Russia 4Laboratory of Molecular and Cellular Toxicology, Department of Industrial Hygiene and
Toxicology, Finnish Institute of Occupational Health, Helsinki, Finland 5Former Department of Molecular Genome Research,
Stockholm University, Stockholm, Sweden 6Laboratory of Molecular Genetics, Cancer Research Institute, Bratislava, Slovak
Republic
(Received 4 November 2003; accepted 20 January 2005)
Abstract
The purpose was to compare the radiation-induced apoptosis in human lymphocytes with DNA-loop relaxation and DNA
damage as a function of radiation dose and time after exposure. Morphological changes were analysed by staining with
fluorescent dyes and apoptotic fragmentation of DNA with conventional agarose gel electrophoresis, pulsed-field gel
electrophoresis (PFGE) and alkaline comet assay. Viability was estimated by trypan blue assay. The levels of protein p53
(TP53) were determined with Western blot. Relaxation of DNA-loops was analysed by the method of anomalous viscosity
time dependence (AVTD) and neutral comet assay. Induction and repair of double-strand breaks (DSB) was studied by
PFGE and by immunostaining of the TP53 binding protein 1 (53BP1). At various time points of apoptosis, there was a linear
dose dependence for all apoptotic end-points up to 1 – 2 Gy followed by a plateau at higher doses. Immediately after
irradiation, relaxation of DNA-loops due to strand breaks was observed. This relaxation had a similar dose – response with
saturation at 2 – 3 Gy. This dose induced approximately one single-strand break (SSB) per 2 Mb of DNA, a value close to
the average size of DNA-loops in resting lymphocytes. Similar saturations in dose – responses for apoptosis and DNA-loop
relaxation were also observed if cells were treated by camptothecin (CPT) or etoposide VP-16, drugs that relax DNA-loops
by induction of SSB and DSB, respectively. The PFGE data showed that the vast majority of DSB were repaired within few
hours after irradiation. However, approximately 1.4 foci/Gy/cell, that corresponded to around 3.5% of initial DSB, remained
in cells even 24 h after irradiation as measured with immunostaining. The probability to produce one or more than one
residual foci per cell was calculated. Radiation at 2 – 3 Gy induced at least one residual 53BP1 focus per cell. The dose –
responses for DNA-loop relaxation, induction of at least one residual 53BP1 foci per cell and apoptosis saturated at 2 – 3 Gy.
The correlation between dose – responses obtained suggested that the DSB in residual foci and relaxation of DNA-loops may
be linked to induction of radiation-induced apoptosis in lymphocytes.
Keywords: Apoptosis, chromatin, human lymphocytes, radiation, repair, DNA-loops.
Introduction
Apoptosis has become recognized as a genetically
controlled mechanism for the regulation of biological
processes such as development, differentiation,
embryogenesis, tissue homeostasis and cell maturation (Raff 1992). It also appears to be of a great
importance as a part of the defence mechanism for
the elimination of deleterious or damaged cells such
as self-reactive lymphocytes, virus-infected or neoplastic cells. Acquisition of resistance to apoptosis
may confer a selective advantage of clonal expansion
upon pre-neoplastic and neoplastic cells. On the
other hand, abnormally increased rates of cell death
may result in degenerative diseases and blood cell
disorders such as Alzheimer’s disease and myelodis-
Correspondence: I. Belyaev, Department of Genetics, Microbiology and Toxicology, Stockholm University, SE-106 91 Stockholm, Sweden.
Tel: 46-8-16-41-08. Fax: 46-8-16-43-15. Email: Igor.Belyaev@gmt.su.se
ISSN 0955-3002 print/ISSN 1362-3095 online # 2005 Taylor & Francis Group Ltd
DOI: 10.1080/09553000500077211
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J. Torudd et al.
plastic syndrome, respectively. Many of the genes
involved in the induction (cell growth associated
genes such as p53 and c-myc) and mediation (the
interleukin-1-converting enzyme (ICE) family of
proteases, the bcl-2 gene family involving B-cell
leukaemia 2 (BCL-2) and B cell associated protein
x (BAX)) of apoptosis have been identified (Wyllie
1995, Shen and White 2001).
Ionizing radiation and many anticancer drugs
induce apoptosis by a TP53-dependent pathway that
is triggered by DNA damage. TP53 functions for the
apoptotic process and for the sensitivity of tumour
cells to radiation have been reviewed (Brown and
Wouters 1999). About 50% of all tumours have
mutations in TP53. Relationships between TP53
status and radiation-induced radioresistance have
been analysed (Russell et al. 1995, Li et al. 2001).
The radioresistant phenotype is often, but not
always, correlated with a decreased rate of apoptosis
(Brown and Wouters 1999). There are also TP53independent processes that are involved in radiationinduced apoptosis (Hara et al. 2004).
It is widely accepted that DNA strand breaks are
responsible for radiation-induced apoptosis. DSB are
assumed to be the most important lesion to induce
apoptosis (Story et al. 1994). The role of SSB in
apoptosis has also been addressed (Tounekti et al.
2001). Although hydrogen peroxide (H2O2) induces
mostly SSB, apoptosis is induced in human lymphocytes upon treatment with H2O2 as analysed by
nuclear morphology and high molecular weight
DNA fragmentation (Marini et al. 1996).
We and others have previously shown that g-rayinduced apoptosis in human lymphocytes saturates
at doses of 1 – 3 Gy (Vral et al. 1998, Belyaev et al.
2001). In previous studies (Vral et al. 1998, Kern et
al. 1999, Belyaev et al. 2001), doses below 5 Gy were
used and apoptosis might hypothetically be increased
at higher doses, especially at the earliest time points
after irradiation. The time points earlier than 24 and
later than 72 h have not previously been investigated.
In the present study, cells were irradiated in the dose
range up to 15 Gy and apoptosis was analysed at 4 h
and up to several days following irradiation. We also
present new data suggesting that apoptosis correlates
with relaxation of DNA-loops caused by SSB or
DSB in response to treatment with the anticancer
drugs camptothecin (CPT) or the etoposide VP-16,
respectively. These drugs are specific inhibitors for
topoisomerases I and II. CPT produces SSB by
stabilizing the topoisomerase I – DNA cleavable
complexes. Similarly, VP-16 produces DSB by
stabilizing the topoisomerase II – DNA cleavable
complexes (Kingma and Osheroff 1998).
Radiation-induced strand breaks are repaired
within a few hours following irradiation. However,
specific slowly repaired or unrepaired DSB may be
responsible for induction of apoptosis. Using PFGE,
it has recently been found that the cellular radiosensitivity measured after delayed plating of 12
human fibroblast lines, derived from the breast
cancer patients, did not correlate with the initial
number of DSB or their repair kinetics, but rather
with the number of residual DSB (Dikomey and
Brammer 2000). In another study (Eastham et al.
2001), only a correlation of borderline significance
was seen between clonogenic survival and residual
DSB in nine human cervix carcinoma cell lines.
However, in these studies DSB and radiosensitivity
were measured at fairly different doses. Cellular
radiosensitivity was usually determined for doses of
up to 8 Gy, while the yield of DSB has been
measured for a dose range of 50 – 200 Gy. For the
PFGE assay, these high doses had to be used in order
to detect residual DSB.
Several proteins involved in DNA repair and DNA
damage signalling such as the tumour suppressor
53BP1, phosphorylated histone H2AX (g-H2AX)
and histone deacetylase 4 (HDAC4) have been
shown to produce discrete foci colocalizing to DSB
(Rogakou et al. 1999, Schultz et al. 2000, Rappold et
al. 2001, Fernandez-Capetillo et al. 2002, Sedelnikova et al. 2002, Kao et al. 2003). The g-H2AX foci
are formed in megabase size domains, presumably
representing condensed globules of DNA-loops
(Rogakou et al. 1999). The 53BP1 protein shares
sequence homology with the checkpoint protein
Rad9 in S. cerevisiae and functions in the same
checkpoint pathway as ATM (ataxia telangiectasia
mutated) (DiTullio et al. 2002). The 53BP1 has a
Myb domain responsible for binding to DNA,
kinetochore-binding domain (KBD) and the Tudor
domain that is able to bind to chromatin (Iwabuchi
et al. 2003). The g-H2AX and 53BP1 proteins are
phosphorylated in response to DNA damage providing a scaffold structure for DSB repair (DiTullio et
al. 2002). According to the current model, this
scaffold functions by recruiting proteins involved in
the repair of DSB (Fernandez-Capetillo et al. 2002,
Iwabuchi et al. 2003, Kao et al. 2003). The scaffold
is organized within a megabase-size chromatin
domain at a site of an actual DNA double-strand
break regardless of the type of repair that is further
involved in processing DSB (Paull et al. 2000). We
studied radiation-induced 53BP1 foci in human cells
of different types: G0 lymphocytes, normal fibroblasts VH-10 and HeLa cells. We have found that
residual foci remained in these cells for a long time
after irradiation, 12 – 24 h, depending on cell type
(Belyaev et al. 2002, Markova et al. 2003). Recent
studies indicated correlation between radiosensitivity
of cells and residual foci (Belyaev et al. 2002,
Iwabuchi et al. 2003, Markova et al. 2003, Rothkamm and Lobrich 2003).
Dose – response in human lymphocytes
Dose dependence for residual 53BP1 foci in
lymphocytes 24 h after irradiation is presented here.
The results obtained suggested that DSB in residual
53BP1 foci and relaxation of DNA-loops might be
linked with induction and execution of radiationinduced apoptosis in lymphocytes.
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changes characteristic for apoptosis, such as chromatin condensation, fragmentation of nuclei and
nuclei shrinkage, were scored by using fluorescence
microscope, three times, 100 counted cells each
time, as previously described (Belyaev et al. 2001).
Anomalous viscosity time dependence (AVTD)
measurements
Materials and methods
Peripheral blood from healthy donors was obtained
from Blodcentralen, Karolinska Hospital, Stockholm.
Experiments
were
performed
with
lymphocytes from different individuals. Lymphocytes
were isolated by density gradient centrifugation in
Ficoll-Paque (Pharmacia LKB Biotechnology, Uppsala, Sweden) according to the manufacturer’s
instructions. The cells were transferred to basal
medium (BM); Roswell Park Memorial Institute
(RPMI) 1640 medium supplemented with 10%
foetal calf serum, 2 mM L-glutamine, 50 IU ml – 1
penicillin, 50 mg ml – 1 streptomycin (ICN Pharmaceuticals, Inc., Costa Mesa, CA, USA). Adherent
monocytes were removed by overnight incubation of
the cell suspension in 170-cm2 Falcon culture flasks
(Becton Dickinson & Co., Franklin Lakes, NJ, USA)
at the cell density of 3 6 106 cells ml – 1. After the preincubation, the cells in suspension were collected by
centrifugation. The cell density was adjusted to 2 – 3
6 106 cells ml – 1 in fresh Basal Medium (BM) and
the lymphocytes were maintained at 5% CO2 and
378C in a humidified incubator (Nunc, Roskilde,
Denmark). The viability of cells was above 95% as
measured with the trypan blue exclusion assay.
DNA-loop relaxation was studied by the AVTD
method. Cell lysis was performed as previously
described with some modifications (Belyaev et al.
1999). Before lysis, the samples were taken from
the cell suspension and diluted with fresh media to
a concentration of 8 6 105 cells ml – 1. Lymphocytes were lysed in 14-mm polyallomer centrifuge
tubes (Beckman Coulter, Inc., Fullerton, CA,
USA) by addition of 3 ml lysis solution (0.25 M
disodium salt of ethylenediaminetetraacetic acid
(Na2EDTA), 2% w/v sarcosyl, 10 mM Tris-base,
pH 7.4) to 0.3 ml cell suspension. The lysates were
kept at 268C for 4 h in darkness before AVTD
measurements. The AVTD in lysates were measured as described previously (Belyaev et al. 1999)
using an AVTD analyser (Archer-Aquarius Ltd,
Moscow, Russia). The AVTD were measured at
the shear rate of 5.6 s – 1 and shear stress of
0.007 N m – 2. For each experimental condition,
AVTD was measured in three replicates. AVTD
parameters were described in detail previously
(Belyaev et al. 1999). Briefly, the AVTD is
characterized by three main parameters: (1) maximum viscosity, measured as normalized maximum
relative viscosity (NRV); (2) area under AVTD,
measured as relative area; and (3) time for
maximum viscosity, measured as relative time. All
these parameters depend on conformation, rigidity
and molecular weight of nucleoids (Belyaev et al.
1999). Normalized maximum relative viscosity is
the most sensitive parameter and usually used to
determine the effects of irradiation.
Irradiation
Western blot
Chemicals
When not otherwise stated, reagent grade chemicals
were obtained from Sigma (St Louis, MO, USA) and
Merck KgaA (Darmstadt, Germany).
Cells
137
The cells were irradiated with
Cs g-rays using
Gammacell 1000 (Atomic Energy of Canada Limited, Ottawa, Canada) source. The dose rate was
10.6 Gy min – 1. After irradiation, the cells were
incubated in a CO2 incubator at 378C.
Fluorescence microscopy
At different time points after exposure, samples from
both irradiated and control cell suspensions were
taken for the assessment of morphological changes.
After staining with fluorescent dyes (acridine orange
and propidium iodide), the cells with morphological
The cells were lysed and proteins were separated by
12% sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE) using Mini-PROTEAN II Electrophoresis Cell according to the
manufacturer’s instruction manual (BioRad, Richmond, CA, USA). Transfer of the proteins onto the
membrane was performed using Mini Trans-Blot
Electrophoretic Transfer Cell (BioRad). Western
blot analysis was performed using the RPN 2108
ECL system according to the manufacturer (Amersham Life Science, Amersham, UK) with some
modifications. Membranes were incubated with the
primary and secondary antibodies, 0.1 mg ml – 1
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J. Torudd et al.
mouse monoclonal anti-TP53 (Ab-6) or 0.2 mg ml – 1
mouse monoclonal anti-TP53 (DO-1), 1 mg ml – 1
mouse monoclonal anti-BCL-2 and 5 mg ml – 1
mouse monoclonal anti-actin antibodies from Calbiochem (Cambridge, MA, USA), Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA, USA), DAKO
(Glostrup, Denmark) and Boehringer Mannheim
(Scandinavia AB, Bromma, Sweden), respectively.
Secondary anti-mouse Ig antibody NA 931 (Amersham) was used at the concentration of 1 mg ml – 1.
Detection of the labelled antibodies was performed
according to the manufacturer manual for ECL
Western blotting detection (Amersham). The membranes were exposed to ECL Hyperfilms
(Amersham) or charge-coupled device (CCD) camera (FUJIFILM Luminescent Image Analyser LAS1000plus). Measurements of integrated optical density (IOD) and area were performed on the films
using Quantiscan (Biosoft, Cambridge, UK) software. Both methods of measurements provided
approximately the same results.
Comet assay
The neutral comet assay as described by Ostling
and Johanson (1984) is sensitive to the loss of
supercoiling produced by single- and double-strand
breaks. The comet assay was performed as
previously described with some modifications
(Klaude et al. 1996). For each slide, 5000 –
10 000 cells were mixed with 150 ml 0.75% lowmelting agarose type VII (Sigma) in phosphatebuffered saline (PBS) held at 378C. The agarose
was spread on ordinary clear-glass slides (Menzel
Superfrost, Braunschweig, Germany) that had been
pretreated with a small amount of agarose and airdried. After solidifying on a chilled plate, the slides
were transferred to the same lysis buffer as for the
AVTD assay and incubated at room temperature
for 4 h in darkness. All subsequent steps were
carried out at room temperature. The slides were
then processed according to either neutral or
alkaline methods as described below:
. Neutral method: slides were incubated in Trisacetate/EDTA buffer (TAE), pH 8.3 for 1 h and
electrophoresis was run at 2 V cm – 1 for 15 min
in TAE buffer.
. Alkaline method: after a 1-h incubation in TAE,
the slides were denatured in 0.03 M NaOH, 1 M
NaCl, 2 mM EDTA and 0.5% sarcosyl for 1 h.
Afterwards, they were rinsed in 0.03 M NaOH,
2 mM EDTA for 1 h and electrophoresed at
0.8 V cm – 1 for 15 min in the same solution. The
slides were then neutralized in 0.4 M Tris-HCl
(pH 7.5), rinsed in distilled water and air dried.
When dry, they were fixed in methanol, stained
.
5 min with 1 mg ml – 1 ethidium bromide and
rinsed 5 min with TAE buffer.
Analysis of tail moments was done with an
Olympus fluorescence microscope, aided by the
Perceptive Instruments Comet Assay V1.03
image analysis system. We also stained with
4’,6-diamidino-2-phenylindole (DAPI) and both
techniques of staining provided similar results.
Analysis of apoptotic cells with extensive fragmentation of DNA was performed using the comet assay as
previously described (Czene et al. 2002). The cells
with highly damaged DNA releasing low molecular
weight fragments of DNA (the visible bright rest of
the head is accompanied with a diffuse but still
distinct tail that is well separated from the head and
wider than the head’s diameter) were counted in
blind to treatment condition.
Pulsed-field gel electrophoresis (PFGE)
PFGE grade agarose, S. cerevisiae, S. pombe, l ladder
and l digest pulse markers and low melting agarose
were purchased from BioRad. A 1 Kb Plus DNA
Ladder was from Gibco BRL, Life Technologies
(Gaithersburg, MD, USA). Proteinase K was purchased from Boehringer Mannheim (Scandinavia
AB, Bromma, Sweden).
The cells were washed twice with and resuspended
in PBS at a density of 0.5 – 1.0 6 107 cell ml – 1. The
suspensions were mixed 1:1 with 1% low melting
point agarose (Sigma). Agarose blocks were prepared
using 100 ml plug moulds. The cells were lysed by
incubation of agarose blocks in NDS (1% lauryl
sarcosyl, 0.5 M EDTA, 10 mM Tris, pH 8.0 adjusted by NaOH) with 0.5 mg ml – 1 proteinase K at
378C for 24 h (Protocol A) or 48 h (Protocols B, C).
After being washed three times with TE buffer
(10 mM Tris, 1 mM EDTA, pH 8.0), blocks were
loaded into the wells in duplicate or triplicate. PFGE
was run either by Protocols A and B for apoptotic
DNA fragmentation or by Protocol C for DSB
measurements (Belyaev and Harms-Ringdahl 2002).
All runs were at 148C in 0.5 6 Tris-borate-EDTA
(TBE) buffer (Maniatis et al. 1982) using a CHEFDR II apparatus (Bio-Rad). Protocol A: 1% agarose
gel, 180 V, run time 22 h and ramped pulse from 10
to 90 s. Protocol B: 1.5% agarose gel, 190 V, run
time was 7 h and pulse time was ramping from 1 to
30 s. In this protocol, apoptotic DNA fragments are
compressed into one compression zone with the
same position on the gels as compressed l digest
markers (Belyaev and Harms-Ringdahl 2002). To
quantify apoptotic fragmentation, different concentrations of the l digest marker of similar size
distribution, 8.3 – 48.5 kb, were embedded in plugs
and run along with apoptotic samples. Calibration
Dose – response in human lymphocytes
curves were obtained based on image analysis and
assuming that each cell has 6 pg DNA. Protocol C
employed the following steps in 0.9% agarose gel: (1)
48 h, at 35 V; (2) 48 h, at 50 V; a 96 h, pulse ramp
overlapped these two steps with pulses decreasing
from 90 to 45 min; and (3) 48 h, at 60 V with pulse
ramp from 45 min to 2 s. On completion, the gels
were stained with 0.5 mg ml – 1 ethidium bromide,
destained and images were acquired at appropriate
saturation using a CCD-camera (Molecular Dynamics). The images were analysed using
Quantiscan (Biosoft) software.
Agarose gel electrophoresis
DNA was extracted with standard phenol-chloroform technique (Maniatis et al. 1982). Conventional
gel electrophoresis was run at 50 V in 1.5%
molecular biology grade agarose (Saveen, Kungsangen, Sweden) with 0.5 mg ml – 1 ethidium bromide in
TAE buffer and room temperature. The gel images
were acquired and analysed in the same way as
described for PFGE (Belyaev and Harms-Ringdahl
2002).
Treatment with CPT and VP-16
Cells were exposed to different concentrations of
CPT or VP-16 (Sigma). Drugs were dissolved in
dimethyl sulfoxide (DMSO). Final concentration of
DMSO in cell cultures during treatment was 0.5%.
Control cells were incubated at the same time with
same concentration of DMSO.
Immunostaining and foci analysis
Anti-53BP1 antibody was kindly provided by Dr T.
Halazonetis, The Wistar Institute, University of
Pennsylvania, Philadelphia, USA. The antibody
recognizes the C-terminal domain of the protein.
The immunostaining was performed according to
Shultz et al. (2000) with some modifications.
Cytospin preparations were fixed in 4% paraformaldehyde at room temperature for 10 min, washed
once with PBS, permeabilized with 0.2% Triton X100 for 5 min at room temperature, washed three
times for 5 min, stained with primary antibody for
1 h, followed by three washes in PBS, incubated with
secondary goat anti-mouse IgG (H + L) antibody
conjugated with fluorescein isothiocyanate (FITC)
or Texas red, washed three times and mounted with
80% glycerol solution in PBS containing 2.5% 1,4diazabicyclo[2.2.2]octane. Bisbenzimide (Hoechst
33258) was added at a concentration of 0.4 mg ml – 1
to the secondary antibody for DNA staining. The
images were recorded on a DAS microscope Leitz
DM RB with a Hamamatsu dual mode cooled CCD
129
camera C4880. In each version of experiment, 30
cells were analysed from each of three to five
randomly selected fields of vision.
Statistical analysis
The data from at least three identical experiments
with cells from different donors were statistically
analysed using Kolmogorov – Smirnov test. Most
data did not fulfil the normal distribution and were
further analysed by the Mann – Whitney U-test, the
Wilcoxon matched pairs signed rank test or regression analysis. Results were considered as significantly
different at p 5 0.05.
Results
Morphological changes during radiation-induced
apoptosis were analysed by staining with acridine
orange and propidium iodide (Figure 1a). No
statistically significant changes in morphology were
observed at 4 h after irradiation (Figure 1a). The
dose dependence after 48 and 72 h were characterized by an initial steep increase in the number of
apoptotic cells up to a dose of 2 – 3 Gy followed by a
plateau up to 15 Gy. The same dose dependence
with saturation at 2 – 3 Gy was observed at later stage
of apoptosis 96 h after irradiation (data not shown).
After 24 h, a slight increase was seen from 5 to 10 Gy
but not as steep as in the beginning of dose –
response.
It has previously been shown that the AVTD peaks
correlate with condensation and fragmentation of
chromatin during apoptosis in human lymphocytes
(Belyaev et al. 2001). In particular, AVTD parameters declined with progression of apoptosis. These
data were confirmed and extended in this study. The
AVTD peaks, e.g. normalized maximum relative
viscosity (NRV), correlated with the number of vital
cells as measured with trypan blue assay from 48 h
up to 168 h after irradiation (Figure 1b). An almost
complete decline in the AVTD parameters was
observed in correlation with cell death at very late
stage of apoptosis, 168 h after irradiation with doses
higher than 4 Gy (Figure 1b).
We also studied the TP53 and BCL-2 protein
levels in cell lysates by Western blot. Actin was used
as an internal control. Variations in levels of actin
were not more than 20% among all samples. A
significant dose-dependent increase in the level of
the TP53 protein was observed after irradiation
(Figure 2a). Dose – response relations for TP53
induction were determined at 4, 24 and 48 h postirradiation (Figure 2b). These dose dependences had
the same shape as those for morphological changes,
i.e. an increase up to 2 Gy followed by a plateau
region between the doses 3 and 15 Gy. In replicated
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J. Torudd et al.
Figure 2. (a) Typical Western blots showing induction of TP53
protein at 4 and 24 h after irradiation of human G0 lymphocytes.
Actin was used as an internal control. (b) Quantifications of
Western blots obtained following 4 h (&), 24 h (.) and 48 h (~)
post-irradiation.
Figure 1. (a) Dose dependence for G0 human lymphocytes with
characteristic apoptotic morphological changes as measured with
fluorescence microscopy at different times after irradiation with 137
Cs g-rays: 4 h (&), 24 h (.), 48 h (~) and 72 h (~). Mean of at
least three experiments and standard deviations are shown here
and in other figures. (b) Condensation and fragmentation of
chromatin during apoptosis as measured by AVTD 48 h (&), 72 h
(*), and 168 h (~) after irradiation and cell viability as measured
with trypan blue assay 48 h (&), 72 h (.), and 168 h (~) postirradiation.
experiments, TP53 induction was rather stable 24 –
48 h post-irradiation. No statistically significant
changes in the BCL-2 protein levels were found
(data not shown).
The fragmentation of DNA in apoptotic cells was
measured with the alkaline comet assay 24 and 48 h
after irradiation. No significant difference between
control and irradiated samples were observed 24 h
after irradiation. The effect of irradiation becomes
significant 48 h after irradiation. The dose dependence had a plateau with saturation at 2 Gy (Figure
3).
Apoptotic fragmentation was also studied by
PFGE. Significant fragmentation of DNA below
50 kb was observed in apoptotic cells at 24 h
(Figure 4), 48, 72 and 96 h after irradiation (data
not shown). In Figure 5, a typical DNA ladder
obtained 96 h after irradiation using conventional
agarose gel electrophoresis along with quantification is shown. The dose – responses for DNA
fragmentation as measured with different techniques and at all time points showed saturation
above 3 Gy.
Relaxation of DNA-loops was observed immediately after irradiation as indicated by the significant
increase in the AVTD peaks. The dose dependence
had a sigmoid shape with a plateau at doses above 2 –
3 Gy (Figure 6a). For neutral comets, the saturation
of dose dependence was similar as for AVTD (Figure
6b). The longest comet tails we observed in neutral
comets were around 325 mm. This length corresponds to approximately 1 Mb of DNA or 2 Mb
loops, assuming 3 bp per nm. Since neutral comets
showed similar dose – response as AVTD we assume
that the saturation in dose – response observed with
these two methods is primarily due to cellular DNAloop organization and not to saturation in DNA
damage.
Dose – response in human lymphocytes
131
Figure 3. Dose dependence for apoptotic cells with extensively
fragmented DNA as measured with alkaline comet assay 48 h after
irradiation. Note that the fraction of apoptotic cells with
extensively fragmented DNA is much lower than the fraction of
cells with morphological changes (10 versus 60%), showing that
extensive fragmentation is a later event in the apoptotic process.
Figure 4. Typical PFGE, protocol A, showing induction of
apoptotic fragmentation below 50 kb. Samples were prepared 4
and 24 h after irradiation of human G0 lymphocytes. Markers: M1
is Lambda digest DNA, 8 – 48.5 kb; M2 is Lambda ladder DNA,
48.5 – 1000 kb.
To compare further the dose dependence for
initial DNA damage and apoptosis, the alkaline
comet assay and PFGE were used. The cells were
irradiated on ice in agarose blocks and lysed
immediately after irradiation. Almost linear dose
dependence was seen with the alkaline comet assay
(Figure 6c). There was no sign of saturation at 2 Gy.
This damage was almost repaired during 2 h after
irradiation (Figure 6d). Induction of DSB was
analysed by PFGE and a dose-dependent increase
of high-molecular weight fragmentation of DNA was
observed (Figure 7). These DSB were repaired
within 3 – 6 h after irradiation and no DNA fragments above 5.7 Mb were observed 24 h after
irradiation (not shown).
Interestingly, significant correlations between
dose dependence for apoptosis and DNA-loop
relaxation were also found following treatment of
cells with either CPT or VP-16. In preliminary
experiments, we used different concentrations of
Figure 5. (a) Typical DNA ladder obtained 96 h after irradiation
with conventional agarose gel electrophoresis. Marker (M) is 1 kb
plus DNA ladder, 100 – 12000 bp. (b) IOD of laddering was
measured in the area within the 100 and 700 kb marker.
CPT and VP-16, and measured AVTD in cell
lysates after different durations of treatment,
10 min to 2 h. For both drugs, DNA-loop relaxation were saturated after 1 h of treatment. CPT
induced complete relaxation of DNA-loops as
measured with AVTD technique at the dose
10 mg ml – 1 (Figure 8a). At the same dose, saturation in apoptosis was observed as measured with
morphological analysis (Figure 8b), apoptotic DNA
fragmentation (Figure 8c), and TP53 induction
(Figure 8d). Dose – responses for VP-16-induced
DNA-loop relaxation and apoptosis as measured
by DNA fragmentation, morphological changes
and viability reached saturation at 100 mg ml – 1
(data are not shown). As shown in Figure 8e and
Table I there was a fairly linear correlation between
relaxation and apoptosis for three different agents.
VP-16 that induces DSB is more efficient than grays that induced mainly SSB with some DSB and
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J. Torudd et al.
Figure 6. Dose dependence of normalized maximal relative viscosity measured with AVTD in lysates of irradiated cells (a) and tail moment
in neutral (b) and alkaline (c) comets. Human lymphocytes were irradiated with 137Cs g-rays at 10.6 Gy min – 1 on ice and lysed immediately
after irradiation. (d) Vast majority of SSB were repaired 2 h post-irradiation as measured with alkaline comets.
Figure 7. Radiation-induced DSB analysed by the PFGE, Protocol
C. Cells were irradiated on ice in agarose blocks and lysed
immediately after irradiation. Marker (M) is S. pombe chromosomal DNA.
g-rays is more efficient than CPT that induces
SSB. The slope was steeper for VP-16 compared
with CPT and g-rays, indicating that loop relaxation is not the only determinant for apoptosis.
To analyse DSB-related foci we used immunostaining with anti-53BP1 antibody (Schultz et al.
2000). Human lymphocytes were irradiated with
3 Gy g-rays and immunostaining was performed
15, 30 min, 4 and 24 h after irradiation. 53BP1
foci formation was maximal 15 – 30 min after
irradiation (Figure 9). At this time after irradiation,
we observed approximately 10 foci/Gy/cell. This
value corresponded to published data (Schultz et
al. 2000). Afterwards, foci disappeared and only
9 + 4 foci/cell were seen 4 h after irradiation with
3 Gy. However, even 24 h after irradiation, a dosedependent number of foci were detected at the
doses of 0.5 – 10 Gy (Figure 10a). These foci were
observed in both apoptotic cells and cells displaying no morphological hallmarks of apoptosis.
Approximately 1.4 foci/Gy/cell was seen in the
cells 24 h after irradiation, this corresponds to 14%
of the foci observed 15 min after irradiation.
However, this value is preliminary as we have not
performed a full analysis of the time kinetics for
foci formation. In addition, the primary foci were
very small in size as compared with the residual
Dose – response in human lymphocytes
133
Figure 8. Dose – response for CPT-induced DNA-loop relaxation (a) and apoptosis (b – d) as measured in human lymphocytes 24 h (&) and
48 h (.) after treatment with CPT. Means and SD are shown from two to four experiments with different donors. (a) Cells were lysed 1 h
after beginning of treatment with CPT and dose dependence for NRV was measured with the AVTD method. (b) Cells were scored for
apoptotic changes. (c) Apoptotic DNA fragmentation was analysed by PFGE, protocol B. (d) The Western blot shown is representative of
induction of TP53 by CPT in one of four experiments. (e) Apoptosis as determined by morphology in dependence on relative relaxation as
determined by AVTD for VP-16 (&, solid line), g-rays (~, short dash) and CPT (., dash). Relative relaxation was determined as the
relaxation at a certain dose relative to the background relaxation.
foci, observed 24 h after irradiation. Thereby, the
number of primary foci might be underestimated.
The number of residual foci constitutes about
3.5% of primary DSB, assuming that g-rays induce
40 DSB/Gy/cell. In Figure 10b, the probability for
one or more residual foci per cell at different doses
134
J. Torudd et al.
Table I. Slopes, intercepts regression and coefficients for the lines plotted in Figure 8e. The significance of the slopes and the dependence of
apoptosis on loop relaxation relative to g-rays are also shown.
g-rays
VP-16
CPT
Slope and intercept
R2
Significance (p)
Slope relative to g-rays
y = 23.0x – 15.3
y = 35.5x – 21.6
y = 19.9x – 19.0
0.9078
0.8972
0.8682
5 0.02
5 0.01
5 0.001
1
1.54
0.86
Figure 9. Formation of 53BP1 foci 15 min after irradiation of human lymphocytes with 3 Gy and residual 53BP1 foci 24 h after irradiation,
as analysed by immunostaining with antibody against 53BP1.
have been calculated using Poisson statistics. Note
that the 3 Gy dose results in at least one residual
foci per cell.
Discussion
A large number of highly conserved genes have been
shown to control the apoptotic process. One of the
most important triggering mechanisms is mediated
by the TP53 protein. The induction of this protein
has been shown during apoptosis in human resting
lymphocytes (Seki et al. 1994). Seki et al. found that
TP53 is induced by irradiation with 0.5 – 15 Gy.
Saturation in dose dependence of TP53 induction
has been observed at 2 – 4 Gy, 4 h post-irradiation.
In our experiments, dose dependence for TP53
induction saturated above 3 Gy at 4, 24 and 48 h
after irradiation. Therefore, our data confirmed and
extended the findings of Seki et al. (1994). No
changes in the level of BCL-2 have been observed
(Seki et al. 1994). In agreement with these data, we
did not observe any changes in the BCL-2 levels
either (not shown).
TP53 is typically induced transiently in most cell
types in response to ionizing radiation. Permanently
induced level of TP53 in G0 human lymphocytes
may be accounted for a rather stable organization of
DNA loops in these cells. The data may suggest that
during the stage of radiation-induced relaxed chromatin in G0 human lymphocytes, TP53 binds to
chromatin and subsequent condensation of chromatin results in a stabilization of bound TP53 (Torudd
et al. 2000).
This study illuminates dose dependence of
radiation-induced apoptosis as measured with
several end-points in resting lymphocytes. The
dose dependences for apoptotic morphological
changes, DNA fragmentation, trypan blue uptake
and TP53 expression saturated above 2 – 3 Gy at
different times after irradiation. Others have
observed similar shapes of dose dependence for
apoptosis in different types of lymphocytes as
measured with various techniques (Hedges and
Hornsey 1978, Filippovich et al. 1982, Sorokina et
al. 1992, Warenius and Down 1995, Vral et al.
1998). The dose dependence for induction of
apoptotic cells was measured by Vral et al.
(1998) 24, 48 and 72 h after g-irradiation of G0
human lymphocytes. Dose – response was characterized by an initial steep increase below 1 Gy,
with a flattening at higher doses towards 5 Gy. In
the same dose range, 1 – 5 Gy, a similar dose –
response was also obtained for morphological
apoptotic changes in our previous study (Belyaev
et al. 2001). Here, we extended the dose range,
used several techniques and analysed apoptosis at
several time points starting with 4 h up to several
days following irradiation. Note that the fraction of
apoptotic cells with extensively fragmented DNA
(Figure 3) is much lower than the fraction of cells
with morphological changes (Figure 1), showing
that extensive fragmentation is a later event in the
apoptotic process. In general, at all time points,
clear saturation in dose – responses was observed
indicating that saturation in dose – response is not a
peculiarity of a specific time point or specific end-
Dose – response in human lymphocytes
Figure 10. Dose – response for residual 53BP1 foci analysed 24 h
after irradiation (a) and for the probability to have one or more
residual foci per cell (b). Foci were scored in irradiated
lymphocytes as described in the Materials and methods. In each
experiment, 30 cells were analysed from each of three to five
randomly selected fields of vision. Means of three experiments
with cells from different donors and standard deviations are
shown. The probability was calculated from the Poisson distribution of residual foci among the cells.
point but a rather general trend of radiationinduced apoptosis in human lymphocytes. Note,
that the increment of dose was 1 Gy in this paper
and slight differences in the dose for saturation
were observed between different time- and endpoints. The presented data may be interpreted as
showing saturation in the range of 2 – 3 Gy.
The role of membrane damage has been considered in induction of apoptosis (Harms-Ringdahl et
al. 1996) but no direct proof has been obtained so far
for low linear energy transfer (LET) radiation.
Radiation-induced apoptosis is commonly believed
to be due to DNA damage. The results presented
herein suggest that sufficient amount of DNA
damage is produced by the dose of 2 – 3 Gy in
135
human lymphocytes to trigger radiation-induced
apoptosis in all cells.
There is a notion that slowly repaired or
unrepaired DSB, as measured 24 h after irradiation, may correlate with clonogenic survival
(Dikomey and Brammer 2000). However, no data
are available to correlate residual DSB with
radiation-induced apoptosis in lymphocytes. In this
study, 53BP1 foci that presumably co-localize to
radiation-induced DSB in different cell types were
analysed (Schultz et al. 2000). According to the
data obtained, one or more residual foci is induced
by doses of 2 – 3 Gy in each cell. DSB in residual
foci may hypothetically represent a sufficient event
for triggering of apoptosis. The majority of DSB
are repaired within few hours post-irradiation.
Localization of DSB in specific areas of chromatin
or complexity of DSB may be important constrains
for the efficiency of the DSB repair. In particular,
two or more DSB might be involved in formation
of residual foci. Such breaks may take longer time
to be repaired and could result in triggering of
apoptotic signal transduction because of the failure
to repair DSB. Other radiation-induced foci may
contain only one, or less complex DSB, which may
be subjected to fast repair and therefore do not
signal for apoptosis.
Residual foci constituted approximately 3.5% of
all radiation-induced foci, assuming that g-rays
induce 40 DSB(foci)/Gy/cell. These foci may be
located to some specific structures of chromatin or
DNA sequences such as nuclear matrix DNA or a
specific fraction of DNA that constitute a minor part
of the genomic DNA. In a recent investigation, Xray-induced 53BP1 foci retained at nuclear foci after
detergent extraction of cells showing that foci localize
in the detergent-insoluble fraction of chromatin
(Iwabuchi et al. 2003). Release of the 53BP1 from
the detergent-insoluble fraction coincided in time
with the extensive 53BP1 focus dispersion (Iwabuchi
et al. 2003). Residual 53BP1 foci remained even
upon extraction of proteins with a standard high saltdetergent protocol for nuclear matrix preparation
(Markova et al. 2003). These data may indicate a
preferential localization of 53BP1 residual foci in the
nuclear matrix.
The comet assay and halo assay have directly
confirmed that the increase in AVTD reflected
relaxation of DNA-loops and that AVTD decrease
was caused by chromatin condensation (Belyaev et
al. 1999, Belyaev et al. 2001). Here, dose – responses
of DNA damage was analysed by the AVTD
technique and comet assay immediately after irradiation. We found similar dose – responses with
saturation at 2 – 3 Gy using both techniques.
Razin and co-workers have described the protocol
for excision of DNA loops by topoisomerase II-
136
J. Torudd et al.
mediated DNA cleavage at matrix attachment sites
(Razin et al. 1995). The cleavage of DNA into
fragments of approximately 2 Mb was observed in
human resting lymphocytes using this protocol in
combination with PFGE (Iarovaia et al. 1995)
(Belyaev et al, unpublished). Assuming an average
loop size of 2 Mb and the length of diploid genome
being 6 6 109 base pairs, the genome of human
lymphocytes will consist of approximately 3000 such
DNA loops. As 1 Gy g-rays induces around 1000
SSB per genome (Ward 1988), most DNA-loops will
be relaxed at the dose of 2 – 3 Gy.
The first data showing plateau in dose – response
as measured by neutral comet assay was described by
inventors of this assay, Östling and Johanson (1984).
Dose – response was measured for L517Y-S cells in
the dose range up to 4 Gy and saturation was
observed at 2 – 3 Gy. We used similar technique for
cell lysis as these authors and our data are comparable. According to Östling and Johanson, DNAloops are stretched out during neutral comet assay
and increases in comet lengths are observed because
of this stretching. At higher doses, DNA fragmentation contributes stronger to increase of comet lengths
as compared with the stretching of DNA-loops and
this increase is usually used for the analysis of DSB
(Banath et al. 1998).
It is attractive to hypothesize that relaxation of
DNA-loops initiate execution of apoptosis in
lymphocytes, facilitating the binding of proteins
such as TP53 to DNA, chromatin or nuclear
matrix. This hypothesis is supported by recent
data that TP53 binds to nuclear matrix in
apoptotic cells of different types including human
lymphocytes (Belyaev et al. 2001, Jiang et al.
2001). The induction of apoptosis at low doses
in some cells does not contradict this hypothesis.
Indeed, some DNA loops containing genes that
are related to induction of apoptosis, will be
relaxed by chance even at low doses. At the dose
that relax all loops, 100% induction of apoptosis
was observed. The data obtained here with VP-16
and CPT support this hypothesis. Despite of
different causes resulting in DNA-loop relaxation
after treatment with CPT and VP-16, SSB or
DSB, respectively, a correlation between apoptosis
and DNA-loop relaxation was observed. The
slope for the correlation was steeper for VP-16
as compared with CPT and g-rays, indicating that
loop relaxation is not the only determinant for
apoptosis (Table I). The slopes were within a
factor of 2, which is quite close for three agents
with such different mechanisms of action. VP-16
that induces DSB is more efficient than g-rays
that induced mainly SSB with some DSB and grays are more efficient than CPT that induces
SSB. For most endpoints, however, DSB is much
more efficient than SSB while in these experiments the difference was a mere 1.8 times.
However, VP-16 and CPT do not induce direct
breaks but protein associated breaks which may
explain the small difference between the two
compounds.
The correlations between dose – responses for
apoptosis, residual foci and DNA-loop relaxation
do not prove that these events are causally related.
Thus, additional data are needed to verify possible
causality. Cells with different radiosensitivities such
as subpopulations of lymphocytes may be further
studied to elaborate this hypothesis. Recent results
(Bakkenist and Kastan 2003) suggest that ionizing
radiation can induce changes in chromatin structure
that activate Ataxia telangiectasia mutated (ATM)
kinase. ATM in turn phosphorylates TP53. Perhaps
this is one of the biochemical links between
chromatin relaxation, apoptosis and residual foci.
Acknowledgements
The authors are grateful to Dr T. Halazonetis for
antibodies to 53BP1, Professor M. Harms-Ringdahl
for valuable discussions and for reading manuscript.
The Swedish Radiation Protection Institute, the
Swedish National Board for Laboratory Animals
and the Swedish Council for Working Life and
Social Research supported this study.
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