Mutagenesis vol. 18 no. 5 pp. 411±416, 2003
DOI: 10.1093/mutage/geg015
Local DNA damage by proton microbeam irradiation induces
poly(ADP-ribose) synthesis in mammalian cells1
Laurence Tartier, Catherine Spenlehauer,
Heidi C.Newman1, Melvyn Folkard1, Kevin M.Prise1,
Barry D.Michael1, Josiane MeÂnissier-de Murcia and
Gilbert de Murcia2
Unite 9003 du CNRS, Laboratoire Conventionne avec le Commissariat aÁ
l'Energie Atomique, Ecole SupeÂrieure de Biotechnologie de Strasbourg,
Boulevard Sebastien Brant, BP 10413, F-67412 Illkirch-Graffenstaden,
France and 1Gray Cancer Institute, PO Box 100, Mount Vernon Hospital,
Northwood, Middlesex HA6 2JR, UK
Cellular recovery from ionizing radiation (IR)-induced
damage involves poly(ADP-ribose) polymerase (PARP-1
and PARP-2) activity, resulting in the induction of a signalling network responsible for the maintenance of genomic integrity. In the present work, a charged particle
microbeam delivering 3.2 MeV protons from a Van de
Graaff accelerator has been used to locally irradiate mammalian cells. We show the immediate response of PARPs
to local irradiation, concomitant with the recruitment of
ATM and Rad51 at sites of DNA damage, both proteins
being involved in DNA strand break repair. We found a
co-localization but no connection between two DNA damage-dependent post-translational modi®cations, namely
poly(ADP-ribosyl)ation of nuclear proteins and phosphorylation of histone H2AX. Both of them, however,
should be considered and used as bona ®de immediate sensitive markers of IR damage in living cells. This technique
thus provides a powerful approach aimed at understanding the interactions between the signals originating from
sites of DNA damage and the subsequent activation of
DNA strand break repair mechanisms
Introduction
Central to pathways that maintain genomic integrity is the
modi®cation of histones and nuclear proteins by ADP-ribose
polymers catalysed by poly(ADP-ribose) polymerases
(PARPs). PARP enzymes now constitute a large family of 18
proteins, encoded by different genes and displaying a conserved catalytic domain (J.C.AmeÂ, C.Spenlehauer and G.de
Murcia, in preparation). Among them, PARP-1 (113 kDa), the
founding member, and PARP-2 (62 kDa) are the sole enzymes
whose catalytic activity is immediately stimulated by DNA
strand breaks, suggesting that they are both involved in the
cellular response to DNA damage (Ame et al., 1999; Schreiber
et al., 2002). At a site of DNA breakage, PARP-1 and PARP-2
catalyse the transfer of the ADP-ribose moiety from the
respiratory coenzyme NAD+ to a limited number of acceptor
proteins involved in chromatin architecture and in DNA
metabolism. Therefore, poly(ADP-ribosyl)ation of nuclear
proteins establishes a molecular link between DNA damage
and chromatin modi®cation and appears as an obligatory step
of a detection/signalling pathway leading ultimately to the
2To
resolution of strand break interruptions (de Murcia and
MeÂnissier-de Murcia, 1994; de Murcia and Shall, 2000).
Recently, the development of novel techniques aimed at
introducing local DNA damage in subnuclear volumes of
cultured cells, using either UVC irradiation through Micropore
®lters (Katsumi et al., 2001; Mone et al., 2001; Volker et al.,
2001) or a UVA laser microbeam (optical scissors) (Rogakou
et al., 1999; Paull et al., 2000), made possible the identi®cation
of early players involved in various DNA repair pathways.
Notably, the concept of a sequential assembly of nucleotide
excision repair (NER) proteins at sites of UVC damage was
proposed (Volker et al., 2001). Moreover, Nelms et al. (1998)
were the ®rst to demonstrate the recruitment of the Mre11/
Nbs1/Rad50 complex involved in the early step of doublestrand break repair using an irradiation mask and ultrasoft Xrays. In the present work, a charged particle microbeam
delivering 3.2 MeV protons from a Van de Graaff accelerator
has been used to locally irradiate mammalian cells. We showed
the immediate activation of nuclear PARP-1 and PARP-2 by
microbeam irradiation concomitant with the recruitment of
ATM and Rad51 at sites of DNA damage, the latter two
proteins being involved in double-strand break repair.
However, no correlation was found between poly(ADPribosyl)ation and phosphorylation of histone H2AX, two
DNA damage-dependent post-translational modi®cations of
nuclear proteins that take place concomitantly at co-localized
sites in response to ionizing radiation (IR).
Materials and methods
Cell culture
HeLa and V79 cells were grown in Dulbecco's modi®ed Eagle's medium
(DMEM) (Gibco), 4.5 g/l glucose medium supplemented with 10% foetal calf
serum, 100 IU/ml penicillin, 100 mg/ml streptomycin, and incubated at 37°C in
an atmosphere of 95% air/5% CO2. Immortalized (3T3) mouse embryonic
®broblasts (MEFs) derived from 13.5 days PARP-1±/± (MeÂnissier de Murcia
et al., 1997) or PARP-2±/± (Schreiber et al., 2002) embryos were maintained in
DMEM, 4.5 g/l glucose medium supplemented with 10% foetal bovine serum
and 0.5% gentamicin.
Irradiation procedure with proton microbeam
The day before irradiation, cells were plated at a density of ~500 cells/dish into
specially designed dishes consisting of a 3 mm thick Mylar base pretreated in
the central area with CellTak adhesive (Becton Dickinson). Cell nuclei were
stained with Hoechst 33342 (Molecular Probes) at a concentration of 1 mM for
~1 h prior to irradiation in order to locate cells on the microbeam dish. The
Hoechst-containing medium was removed at the time of irradiation and
replaced by fresh 20 mM HEPES-buffered medium.
For irradiation, dishes were placed in a maintained atmosphere (4±8°C)
produced by a refrigerated gas ¯ow of 95% air/5% CO2 throughout the
procedure. Dishes were precooled 10 min prior to cell identi®cation. This
operation consists of the acquisition of overlapping frames to result in a
complete picture of all the ¯uorescent objects present in a 7 3 7 mm region.
Recorded cellular locations were revisited at the time of irradiation with the
microbeam targeting the centre of each cell nucleus. Over 99% of the particles
are delivered within 2 mm and 90% within 1 mm of the targeted location.
Irradiation is performed using the Gray Cancer Institute Charged Particle
Microbeam, delivering 3.2 MeV protons from a Van de Graaff accelerator (for
whom correspondence should be addressed. Tel: +33 3 9024 4707; Fax: +33 3 9024 4686; Email: [email protected]
Mutagenesis vol. 18 no. 5 ã UK Environmental Mutagen Society 2003; all rights reserved.
411
L.Tartier et al.
Fig. 1. (A) Detection of locally induced DNA damage in various cell types with microbeam irradiation of 20 Gy (a and b) or whole cell X-ray irradiation with
20 Gy (c). The damaged area was detected by immuno¯uorescent labelling of poly(ADP-ribose) synthesized in response to DNA damage. (b and e) A V79
nucleus targeted at two separate nuclear locations by a 20 Gy proton microbeam. (d±l) Merged images of damaged nuclei stained with DAPI. (B) Mean values
of ¯uorescence from poly(ADP-ribose) foci quanti®ed with Quantity One software (Bio-Rad) following irradiation of HeLa nuclei with doses ranging from 5 to
20 Gy protons (g±l). Bars indicate10 mm.
the microbeam description and extended procedures see Folkard et al., 1997).
Fifteen minutes were needed to scan the dish and locate about 300±500 target
cells which were irradiated at a speed of ~2 s/cell when delivering 600 protons.
Fifty per cent of the cells from a dish were irradiated and the unirradiated
second half was used as a control. Controls and irradiated microbeam dishes
were incubated for 10 min at 37°C before ®xing. Irradiated dishes reached a
temperature of 37°C in <3 min. Control experiments aimed at producing
412
uniform irradiation of cells were performed using conventional 240 kV X-rays,
with cells irradiated on similar dishes.
Dosimetry
The energy deposited by a proton track is dependent on the thickness of the
nucleus and its diameter. These parameters were measured by 2-photon
microscopy using Hoechst (50 mM) stained cell nuclei and Rhodamine 123
DNA damage induces poly(ADP-ribose) synthesis
Fig. 2. Detection of poly(ADP-ribose) synthesis in wild-type (a and d), PARP-1-de®cient (b and e) and PARP-2-de®cient cell lines (c and f) following a
microbeam irradiation of 20 Gy. (d±f) Merged images of damaged nuclei stained with DAPI. Bar indicates 10 mm.
(50 mM) as a cytosol stain. Measurements obtained for HeLa cells gave an
average nucleus thickness of 7.15 mm with an average nuclear area of 100 mm2.
For 3.2 MeV energy protons this gives a mean of 28 protons/Gy (20 Gy equates
to 560 particles). For V79 cells, the mean nucleus thickness is 6 mm with a
nuclear area of 140 mm2. For 3.2 MeV protons this equates to ~45 particles/Gy
(20 Gy equates to 900 particles).
Immuno¯uorescence
Cells were ®xed in an ice-cold mixture of methanol/acetone (50:50 v/v) for 10
min at 4°C. Cells were washed three times with phosphate-buffered saline
(PBS) supplemented with 0.1% Triton X-100, blocked with 0.2% non-fat milk
in PBS/0.1% Triton X-100 (v/v) and incubated overnight at 4°C with either
monoclonal anti-poly(ADP-ribose) antibody (10H) at 1:200 dilution (a gift
from Dr T.Sugimura), polyclonal anti-poly(ADP-ribose) at 1:1500 (Biomol,
Plymouth Meeting, PA), polyclonal anti-gH2AX (Trevigen, Gaithersburg, MO)
at 1:2000 or polyclonal anti-ATM (Santa-Cruz Biotechnology, Santa Cruz,
CA) at 1:1000. After three washes, cells were incubated for 1 h at room
temperature with the appropriate conjugated secondary antibodies to provide
the appropriate combination of species speci®city (goat anti-rabbit or antimouse) and colour discrimination (Alexa-¯uor 488 or Alexa-¯uor 568;
Molecular Probes, Leiden, The Netherlands). Cells were counterstained with
4¢,6-diamidino-2-phenylindole (DAPI) and mounted in MOWIOL.
Immuno¯uorescence was evaluated using either an Axioplan (Zeiss) or a
DMRA2 (Leica) microscope, equipped with a Olympus DP50 CCD camera.
Measurement of ¯uorescence intensity was performed using Quantity One
software (Bio-Rad); 20 cells were scored for each IR dose.
Western blot analysis
For immunodetection, blots were incubated with mouse anti-PARP-1 (EGT69,
1/10 000), af®nity puri®ed anti-PARP-2 (Ab175, 1/1000), anti-g-H2AX (1/
1000), anti-poly(ADP-ribose) (1/1000) or anti-actin (1/3000) (Sigma, St Louis,
MO). Blots where then probed with horseradish peroxidase-coupled secondary
antibodies (goat anti-rabbit and rabbit anti-mouse 1/20 000 (Sigma, St Louis,
MO) and immunoreactivity was detected by enhanced chemiluminescence
(NEN, Boston, MA).
Results
Poly(ADP)ribose synthesis following proton microbeam
irradiation: in situ visualization and dose dependency
The microbeam facility which has been developed at the Gray
Cancer Institute allows in vitro irradiation with charged
particles of distinct cell compartments with a 2 mm beam
spot. Previous studies using this microbeam in bystander
experiments have shown its ability to selectively irradiate a
limited number of cell nuclei (Prise et al., 1998; Belyakov et al.,
2001). In the present experiments, microbeam irradiation is
applied as a new approach aimed at characterizing the
interactions between IR-induced DNA damage and damage
response proteins. For most experiments, HeLa cells were
used, but similar results were obtained with V79 cells and
MEF-derived cell lines. Irradiation was performed on cells
grown on special microbeam dishes, which were precooled to
6°C before irradiation. Cells were ®xed 10 min later and then
immunostained with different antibodies.
HeLa and V79 cells were subjected to microbeam irradiation
with a number of protons equivalent to a dose of 20 Gy/
nucleus. Depending on the software used for the cell identi®cation procedure, cell nuclei were hit at one or more separate
locations by the proton microbeam. The results displayed in
Figure 1Aa and b show the poly(ADP-ribose) signal restricted
to a localized area with a diameter comparable to the beam
size. This is visualized as bright green foci with either
monoclonal (Figure 1A) or polyclonal anti-poly(ADP-ribose)
antibodies (data not shown) 10 min after IR. The majority of
ADP-ribose polymer foci had been removed between 30 and 60
min after IR due to polymer degradation catalysed by the
poly(ADP-ribose) glycohydrolase (PARG). For a given dose
delivered to each cell within a dish, the intensity of polymer
foci are variable and may re¯ect differences in cell cycle
progression. For comparison, poly(ADP-ribose) synthesis in
response to whole cell irradiation of 3T3 cells with 20 Gy Xrays is shown in Figure 1Ac. No poly(ADP-ribose) immunostaining could be detected when cells were pretreated with 10
mM 3-aminobenzamide, a PARP inhibitor, prior to irradiation
(data not shown).
Individual HeLa cell nuclei were exposed to local IR doses
ranging from 5 to 20 Gy (Figure 1B). After 5 Gy irradiation,
only a few cells with foci of weak intensity were observed.
Irradiation with 10 Gy was enough to obtain detectable foci of
variable intensities in most irradiated cells and cell types. The
mean value of intensity of the foci increases with dose and is
maximum at 20 Gy, the dose that we used for all subsequent
experiments. The large error bars re¯ect the high variability of
foci intensities for a given dose (Figure 1Bl).
413
L.Tartier et al.
Fig. 3. Recruitment of DNA repair proteins on damaged sites following 20 Gy proton irradiation. Recruitment of Rad51 protein (a) to poly(ADP-ribose) foci
(e) in V79 cells. ATM (b) is accumulated at DNA damage loci visualized with the anti-poly(ADP-ribose) antibodies (f). (i) is a merge of (a and e), (j) is a
merge of (b and f). Co-localization of phosphorylated histone H2AX (g-H2AX) (c) and poly(ADP-ribose) (g) on DNA damaged sites of 3T3 cells irradiated
with 20 Gy delivered by a microbeam. Whole cell radiation of HeLa cells with 20 Gy of X-ray induces g-H2AX (d) and poly(ADP-ribose) (h) within 10 min.
(k) and (l) are merged images of (c and g) and (d and h) respectively. Bars indicate 10 mm.
PARP-1 knockout cell lines are de®cient in localized IR
response
Given that PARP-1 and PARP-2 are to date the sole known
members of the PARP family whose activity is stimulated in
response to DNA breaks, we sought to visualize microbeaminduced poly(ADP-ribose) synthesis in 3T3 cells selectively
de®cient in one of these two enzymes. As shown in Figure 2,
wild-type cells (Figure 2a) and PARP-2-de®cient cells
(Figure 2c) display comparable immuno¯uorescence signals
corresponding to microbeam-induced poly(ADP-ribose) synthesis. In contrast, no signal was detectable in PARP-1
knockout cells (Figure 2c) under the same radiation conditions
(20 Gy), thus con®rming that PARP-1 is the main enzyme
responsible for the DNA damage-dependent synthesis of
poly(ADP-ribose) (Schreiber et al., 2002).
Recruitment of DSB repair proteins at DNA damage sites
To test whether DNA strand breaks produced locally by
microbeam irradiation of cells would induce the recruitment of
strand break signalling and repair enzymes at DNA damage
sites, we performed double immuno¯uorescence experiments
using polyclonal antibodies against Rad51 or ATM. As shown
in Figure 3a, Rad51, a eukaryotic RecA homologue that plays a
central role in homologous recombinational repair of doublestrand breaks (Daboussi et al., 2002), formed bright foci that
coincide with poly(ADP-ribose) synthesis (Figure 3e). A
similar result was obtained when an antibody against human
ATM (Figure 3b), a phosphatidylinositol 3-kinase (PI3K)
414
involved in signalling and repair of double-strand breaks
(Kastan et al., 2001; Shiloh and Kastan, 2001), was used in coimmunostaining with anti-poly(ADP-ribose) (Figure 3f).
Histone H2AX, one of the three types of conserved histones
H2A, is rapidly phosphorylated at Ser139 in response to DNA
strand breaks (Rogakou et al., 1999). ATM has been shown to
be the major kinase responsible for this DNA damagedependent modi®cation (Burma et al., 2001). As g-H2AX,
the speci®c phosphorylated form of histone H2AX, forms foci
within seconds of DNA damage in¯iction, we therefore looked
at the distribution of g-H2AX foci versus poly(ADP-ribose)
following microbeam radiation. Figure 3c shows the location
of g-H2AX at DNA damage sites. Immunostaining with anti-gH2AX antibody overlapping with poly(ADP)ribose foci was
seen in damaged cells but was not affected by the presence of
10 mM 3-aminobenzamide, a PARP inhibitor (not shown).
Under these conditions g-H2AX foci remained in the localized
area for up to 6 h after IR (not shown), most probably as a
consequence of DNA break persistence due to PARP inhibition. For comparison, whole cell IR with 20 Gy X-rays gave
intense signals with both an anti-g-H2AX antibody (Figure 3d)
and an anti-poly(ADP-ribose) antibody (Figure 3h).
The concomitance of both poly(ADP-ribose) synthesis and
g-H2AX phosphorylation immediately following cell irradiation prompted us to investigate the existence of possible crosstalk between these two post-translational modi®cations of
nuclear proteins induced by DNA strand breaks. For that
purpose, 3T3 cells from the three genotypes, PARP+/+,
DNA damage induces poly(ADP-ribose) synthesis
Fig. 4. Induction of poly(ADP-ribosyl)ation and phosphorylation of H2AX in
wild-type, PARP-1±/± and PARP-2±/± 3T3 cells, 30 min following 20 Gy
irradiation. Each cell line was characterized in terms of PARP-1 and PARP-2
content. Anti-actin was used for loading control.
PARP-1±/± and PARP-2±/±, were globally irradiated with a dose
of 20 Gy. Thirty minutes later, crude cellular extracts were
prepared and analysed by western blotting. As displayed in
Figure 4, the formation of g-H2AX was induced in irradiated
cells whatever the PARP status, whereas polymers of ADPribose were detected mainly in extracts taken from wild-type or
PARP-2 knockout cells, in agreement with the immuno¯uorescence data (Figure 2).
Taken together, these results con®rm that local DNA
damage induced by microbeam radiation triggers several
independent pathways critical for the recruitment of key
factors involved in strand break repair and DNA damage
signalling.
Discussion
X-ray partial volume irradiation of mammalian cells through a
gridded shield demonstrated for the ®rst time the recruitment of
the DNA repair complex Mre11/Rad50/Nbs1 to the sites of
DNA damage as early as 30 min after irradiation (Nelms et al.,
1998). An alternative method using pulsed microbeam (UVA)
laser irradiation in the presence of a photosensitizer was
recently developed to induce local DNA damage (Rogakou
et al., 1999), which in turn triggers the accumulation of repair
proteins, including Rad50, Rad51 and BRCA1 at foci primarily
marked by g-H2AX (Paull et al., 2000). In the present work, a
charged particle microbeam irradiator system has been
developed to induce local DNA damage at the single cell
level. The increase in interest in electron and charged particle
microbeams over the past decade was not only driven by an
interest in low dose IR risk from, for example, radon exposure,
but also by mechanistic studies of cell±cell interactions after
localized IR, such as bystander effects. Another application of
this promising technology is set up in this work, to dissect the
early events that take place in the nucleus of a living cell
injured with a given dose of protons.
Our results point to poly(ADP-ribose) synthesis that immediately follows irradiation. This can be considered as one of the
very early cell responses. Analysis of mutant cells de®cient in
either PARP-1 or PARP-2 revealed that the former is the major
enzyme that massively reacts to DNA breaks, in agreement
Fig. 5. Schematic representation of the DNA strand breaks signalling and
processing pathway. The pathway is triggered by IR resulting in single- and
double-strand breaks that immediately initiate two post-translational
modi®cations of histones and nuclear proteins catalysed by PARP-1/-2 and
ATM. Poly(ADP-ribosyl)ation of histones H1 and H2B increases chromatin
accessibility at the actual site of DNA damage (Poirier et al., 1982; de
Murcia et al., 1988), thus promoting DNA repair. Phosphorylation of histone
H2AX by ATM occurs at or near the double-strand break and is required for
the phosphorylation of 53BP1 that participates in nuclear foci organization
and the subsequent recruitment of several ATM downstream targets
(Fernandez-Capetillo et al., 2002).
with our previous work (Ame et al., 1999; Schreiber et al.,
2002). Locally synthesized polymers of ADP-ribose most
likely display multiple functions at the nucleus level. Firstly, it
may act as an immediate signalling molecule translating the
occurrence of breaks into a massive local synthesis of
negatively charged polymers at the expense of the cellular
energy pool. Secondly, these short-lived polymers in turn play
a role in the recruitment of DNA repair factors like XRCC1
(Masson et al., 1998) and in the necessary conformational
change in chromatin that takes place during repair.
We found that in response to IR-induced damage, local
poly(ADP-ribose) synthesis is accompanied within minutes by
the recruitment of ATM, which plays a crucial role in the rapid
induction of multiple signalling pathways: repair of DNA
damage and activation of cell cycle checkpoints (Kastan et al.,
2001). Rad51, a key actor in the homologous recombination
repair pathway that was shown to be recruited to double-strand
breaks (Tashiro et al., 2000), also rapidly accumulated at
poly(ADP-ribose) foci. More work will be necessary to
establish the spatial and temporal interactions between various
damage signalling molecules and repair enzymes involved in
the IR response. Our results point to a co-localization but
absence of connection between poly(ADP-ribosyl)ation of
nuclear proteins, especially histones, and phosphorylation of
histone H2AX, two post-translational modi®cations of nuclear
proteins induced by IR. Interestingly, the simultaneous
inactivation of either PARP-1 and ATM (Menisser-de Murcia
et al., 2001) or PARP-2 and ATM (A.Huber and J.MeÂnissier-de
Murcia, in preparation) in mice led to early embryonic
415
L.Tartier et al.
lethality, suggesting an absolute necessity to maintain at least
one of these two important pathways, especially during early
development. Thus, both modi®cations appear to constitute a
part of the histone code (Strahl and Allis, 2000; Jenuwein and
Allis, 2001) translating the occurrence of a break into
molecular signals emanating from the damaged chromatin to
facilitate DNA repair and cell survival (Figure 5). However,
differences in their respective kinetics, 30±60 min for
poly(ADP-ribosyl)ation versus several hours for g-H2AX
(Paull et al., 2000), re¯ect different functions in the cell
response to IR. Poly(ADP-ribosyl)ation is mostly associated
with single-strand break repair whereas the ATM/H2AX
pathway is activated in the presence of double-strand breaks,
which are known to be repaired more slowly. In any case, both
should be considered and used as bona ®de sensitive and
immediate markers of IR-induced damage in living cells.
Acknowledgements
The authors are grateful to Dr V.Favaudon for critical reading of the
manuscript, to Simon Ameer-Beg for assistance with the 2-photon measurements and to Dr Sugimura for the 10H anti-poly(ADP-ribose) antibody. This
work was supported by funds from CNRS, Association pour la Recherche
Contre le Cancer, Electricite de France, Ligue Nationale Contre le Cancer,
Commissariat aÁ l'Energie Atomique and Cancer Research UK.
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