MCB Accepts, published online ahead of print on 10 March 2014 Mol. Cell. Biol. doi:10.1128/MCB.01345-13 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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The tumor suppressor PML specifically accumulates at RPA/Rad51 containing DNA
2
damage repair foci but is non-essential for DNA damage-induced fibroblast senescence
3 4
Sandra Müncha,*1, Stefanie Weidtkamp-Petersa,*2, Karolin Klementa,*3, Paulius
5
Grigaraviciusa,*4, Shamci Monajembashia, Paolo Salomonib, Pier Paolo Pandolfic, Klaus
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Weißhartd, and Peter Hemmericha#
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Leibniz-Institute For Age Research, Jena, Germanya; University College London, UCL Cancer
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Institute, London, Great Britainb; Cancer Research Institute, Beth Israel Deaconess Cancer
10
Center, Department of Medicine and Pathology, Beth Israel Deaconess
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Medical Center, Harvard Medical School, Boston, MA 02215, USAc; Carl Zeiss Microscopy
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GmbH, BioSciences Division, Jena, Germanyd.
13 14
Running Head: PML is dispensable for DNA damage-induced senescence
15 16
#Address correspondence to Peter Hemmerich,
[email protected]
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*1Present address: Medical Research Institute, Ninewells Hospital & Medical School, Dundee,
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Scotland, UK; *2Present address: 3Center for Advanced Imaging, University of Düsseldorf,
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Germany; *3Present address: Department of Biochemistry & Molecular Biology, Faculty of
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Medicine, University of Calgary, AB, Canada; *4Present address: German Cancer Research
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Center, DNA-Repair and CNS diseases, Heidelberg, Germany.
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word count for Materials and Methods section: 1,352
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combined word count for introduction, results, and discussion sections: 4,975
1
1
Abstract
2
The promyelocytic leukemia (PML) tumor suppressor has been functionally implicated in the
3
DNA damage response and cellular senescence. Direct evidence for such a role based on PML
4
knockdown or knockout approaches is still lacking. We have therefore analyzed the irradiation-
5
induced DNA damage response and cellular senescence in human and mouse fibroblasts lacking
6
PML. Our data show that PML nuclear bodies (PML NBs) non-randomly associate with
7
persistent DNA damage foci in unperturbed human skin and in high-dose irradiated cell culture
8
systems. PML bodies do not associate with transient γH2AX foci after low dose γ-irradiation.
9
Superresolution microscopy reveals that all PML bodies within a nucleus are engaged at Rad51
10
and RPA containing repair foci during ongoing DNA repair. The lack of PML (i) does not
11
majorly affect the DNA damage response, (ii) does not alter the efficiency of senescence
12
induction after DNA damage and (iii) does not affect the proliferative potential of primary mouse
13
embryonic fibroblasts during serial passaging. Thus, while PML NBs specifically accumulate at
14
Rad51/RPA containing lesions and senescence-derived persistent DNA damage foci, they are not
15
essential for DNA damage-induced and replicative senescence of human and murine fibroblasts.
16 17 18
2
1
Introduction
2
Cellular senescence was first observed by Hayflick and Moorhead in primary human cell
3
culture systems in vitro. The major hallmark of senescent cells is a stable cell cycle arrest after a
4
finite number of in vitro duplications (1). Senescence can be triggered by telomere shortening
5
and non-telomeric pathways including oncogene activation and persistent DNA damage. The
6
pathways involving p53 and p21 as well as pRB and p16 are essential for a functional telomeric
7
and non-telomeric DNA damage response (DDR) (2). It is now firmly established that cellular
8
senescence can act in vivo as an important barrier against cancer progression but also contributes
9
to aging-related tissue pathologies (3).
10 11
The finding that senescence-associated DNA damage foci (SDF) of telomeric and non-
12
telomeric origin accumulate in senescing cells indicated DNA double-strand breaks (DSBs) as a
13
critical factor in the senescence and aging process (4-7). Stress-induced premature senescence
14
(SIPS) is considered to be elicited by widespread non-telomeric DNA damage in cells exposed to
15
genotoxic stress (8). Regardless of the origin, SDF display as persistent DNA damage foci at
16
which many known DDR factors such as γH2AX, ATM, ATR, 53BP1, and the MRN complex
17
are accumulated (9). The accumulation of persistent DNA damage foci is a common process in
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mammalian aging in vivo and in cell culture systems (7, 10-14).
19
Transient foci represent sites of successful DSB rejoining, whereas persistent (late) foci contain
20
unrepairable DSBs (7, 15, 16). The two types of foci can also be distinguished by their DNA
21
repair protein content (7) and spatial association with promyelocytic leukemia (PML) nuclear
22
bodies (15, 17-19). More recently it was shown that persistent foci lack evidence of DNA
23
synthesis, single-stranded DNA and homologous recombination repair (19).
3
1 2
Promyelocytic leukemia (PML) NBs are spherical protein accumulations present in most
3
mammalian cell nuclei (20). Their major structural component is PML. Some factors interacting
4
with PML are linked to the DDR, therefore PML bodies are proposed to be involved in DNA
5
repair, apoptosis, cellular senescence and tumor suppression (21-25). PML NBs were also found
6
in spatial proximity to DNA single-strand breaks (SSBs) and DSBs (17, 26, 27). This suggests
7
that PML NBs could serve as DNA damage sensors, DNA repair compartment and physical sites
8
where DNA repair activities and/or cell cycle checkpoint pathways are coordinated and
9
monitored (17, 28). PML protein levels and the number of NBs are elevated when cells
10 11
encounter stress, e.g. after DNA damage (28-30) and during senescence induction (31, 32). Overexpression of PML protein isoform IV induces senescence in primary human and
12
murine fibroblasts and this process is dependent on p53 and pRb (32-34). The underlying
13
mechanism involves a PML VI-mediated inhibition of E2F target gene expression, followed by a
14
proliferation block, DNA damage induction and senescence (35). PML-depleted cells show
15
alterations in their response to DNA damage and senescence induction. Certain cell types from
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PML knockout (ko) mice showed a decreased apoptosis rate in response to multiple stimuli
17
including γ-irradiation (γ-IR), UV and DNA-damaging agents (36-41). PML knockout and
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knockdown murine embryonic fibroblasts are resistant to Ras-induced senescence (40, 42). Also
19
the activation of p53 is reduced in PML-depleted mouse and human cells (37, 39, 40, 42, 43).
20 21
Despite all these data the precise function of PML in the DNA damage response is not
22
fully understood. We therefore analyzed the DNA damage response and cellular senescence in
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the presence or absence of endogenous PML. Surprisingly, ablation of PML did not alter the
4
1
cellular DNA damage response or senescence induction in primary human or mouse embryonic
2
fibroblasts. These observations reveal a non-essential role for PML in fibroblast senescence.
3 4 5 6
Materials & Methods
7
Cell culture
8 9
WI-38 cells were obtained from the American Tissue Culture Collection (ATCC) and were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal
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calf serum (FCS) in a 10% CO2 atmosphere at 37°C. Primary human foreskin fibroblasts (HFFs)
11
with a stable, short interfering RNA-mediated knockdown of PML as well as the two control cell
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lines were a kind gift of Thomas Stamminger and Nina Tavalai, Erlangen, Germany. The cells
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were generated as described in (44). Primary MEFs were isolated at day E13.5 and cultured in
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Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS and 200 mM L-
15
Glutamin in a 5% CO2 atmosphere at 37°C.
16
γ-Irradiation and drug treatment
17
Cells were γ-irradiated (137Cesium, 1Gy/min, Gammacell GC40, Nordion, Ottawa,
18
Canada) or UV-A-irradiated (see Suppl. Information). For drug-induced senescence cells were
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treated constantly for 6 days with 50 µM 5-bromodeoxyuridine (BrdU) and10 µM distamycin A
20
(DMA) as described previously (45).
21
UV-A microbeam induced DNA damage
22 23
For laser damage induction, the pulsed UV-A laser was coupled into a confocal laser scanning microscope (LSM 510) via the epifluorescence illumination path. A laser-microbeam
5
1
was focused into the middle of the field of view by a 100X, NA 1.3 Plan Neofluar oil immersion
2
objective (Zeiss). The UV-A laser was a frequency tripled Nd:YLF laser (Spectra Physics)
3
delivering 20 ns duration pulses at 350 nm with user-defined energies from 1µJ to 200 µJ at user
4
defined repetition rates 1 Hz -1000 Hz. Before entering the microscope laser pulse energy was
5
reduced by 80% with the gradient position dependent attenuator (Laseroptik). The cells were
6
irradiated in a quadrangular 500 µm x 535 µm area by moving the motorised x,y table, which
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was driven by an MCU 26 controller (Zeiss) at 1470 µm/s speed. In this case we used 350 Hz
8
repetition rate. By selected irradiation options approximately 100 cells were irradiated in 25
9
seconds with single pulses hitting the cell nucleus every 4 µm. No sensitisation was used.
10 11 12
Indirect immunofluorescence and Western blot analysis Cells grown on glass coverslips were fixed in 4% formaldehyde for 10 min and
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permeabilized with 0.25% Triton X-100 for 3 min. Human skin samples were a kind gift of
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Johannes Norgauer, Jena, Germany, and have been described previously (45). 12 µm sections
15
were fixed in 4% formaldehyde for 10 min and permeabilised with 0.25% Triton X-100 for 10
16
min. After the staining, coverslips were mounted in Prolong Gold antifade mounting medium
17
with DAPI (Invitrogen, Karlsruhe, Germany). For Westernblotting whole-cell extracts were
18
prepared by incubation at 100°C for 10 minutes, separated by SDS-PAGE and transferred to a
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nitrocellulose membrane. Quantification of band intensities was carried out with the Metamorph
20
software (MDS Analytical Technologies, Sunnyvale, USA).
21
Antibodies
22 23
Primary antibodies used in murine and human cells: anti-phospho-H2AX (Ser139) (γH2AX) clone JBW301 (Millipore, Billerica, USA), anti-β-actin A5441, anti-α-tubulin T9026 6
1
(both: Sigma, Taufkirchen, Germany). Primary antibodies used in human cells: anti-PML, anti-
2
SP100 (both: Peptide Speciality Laboratories, Heidelberg, Germany), anti-Mdc1 NB100-395,
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anti-53BP1 NB100-305, anti-phospho-Nbs1 (Ser343) NB100-284 (all: Novus Biologicals,
4
Littleton, USA), anti-TRF2 (IMG-124, Imgenex, San Diego, USA), anti-phospho-ATM
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(Ser1981) ab81292, anti-Ki-67 ab15580 (both: Abcam, Cambridge, UK), anti-p16 BD554079,
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anti-pRb BD554136 (both: BD Pharmingen, Heidelberg, Germany), anti-p21 H-164 (Santa Cruz
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Biotechnology, Santa Cruz, USA), anti-p53 OP43T (Millipore, Billerica, USA); anti-RPA-p34
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(Ab-1, Thermo Scientific, Waltham, USA); anti-Rad51 (3C10 mAB, Millipore; Billerica, USA).
9
Primary antibodies used in murine cells: anti-PML clone 36.1-104 (Millipore, Billerica, USA),
10
anti-53BP1 (Bethyl Laboratories, Montgomery, USA). Cy2-, Cy3- or Cy5-fluorescence-labeled
11
secondary antibodies as well as HRP-labeled secondary antibodies were obtained from Jackson
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Immnuo Research, Newmarket, UK.
13
Senescence-associated β-galactosidase staining (SA-βgal)
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Cells grown on glass coverslips were fixed in 4% formaldehyde for 5 minutes and stained
15
for senescence-associated β-galactosidase activity as described previously (46). Cells were
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quantified using an Axioplan2 microscope with a 63x Plan-Apochromat oil objective (Carl Zeiss,
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Jena, Germany).
18
Microscopy and foci quantification
19
Images were aquired with a Zeiss LSM 510 laser scanning confocal device attached to an
20
Axioplan 2 microscope using a 63x Plan-Apochromat oil objective (Carl Zeiss, Jena, Germany).
21
For quantification of γH2AX foci and PML NBs as well as association events optical sections
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(~0.5 µm) of the whole cell nucleus were taken and reduced to one plane by maximum
23
projection. For the quantification with the open source softwar ImageJ and customized macros 7
1
images were processed as described previously (47). Association events were scored with the
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colocalization macro available on the ImageJ website (rsbweb.nih.gov/ij/) and were defined as
3
the overlap of PML and γH2AX signals in at least 5 pixels.
4 5
Structured Illumination Microscopy (SIM)
6
SIM was performed on an ELYRA S.1 system from Carl Zeiss using a C-Apo 63x oil objective
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with a numerical aperture of 1.4. An Andor iXon 885 EMCCD camara served as the detector.
8
The gain of the camera was set to a maximum of 10 to maintain a high dynamic range. For
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multicolor acquisition the sample was sequentially imaged using grids matched to the different
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wavelengths. Five rotations were used for a maximum homogeneous resolution enhancement in
11
the lateral plane. For 3D-SIM, stacks with a step size of 120 nm were recorded. For
12
reconstruction of SIM images the SIM processing tool of the built in ZEN software was used in
13
automatic mode. Refinement of the reconstruction was done manually by adapting the noise
14
filter. Chromatic aberrations of the optical system were determined and corrected with the in-
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built channel alignment tool of the ZEN 2011 software using TetraSpeckTM 0.1 µm beads (Life
16
Technologies) as fiducial markers.
17 18
Theoretical association probability
19
To calculate the theoretical probability for a colocalization event between sphere-shaped objects
20
(PML bodies and IRIF) in the nucleus, the volume of the nucleus and the number and volume of
21
the objects were taken into account. These data were determined from 3-D image stacks of
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immunofluorescence experiments. In case of a colocalization (or association) event of an IRIF
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and a PML body, the distance between the centers of the two colocalizing distinct spheres within
8
1
the nucleus is equal or less than the sum of the sphere's radii, d < rg + rp, where rg is the radius of
2
the genomic locus and rp is the radius of a PML body. Considering one single nucleus of radius R
3
with m PML bodies of radius rp each, and n IRIF with radius rg each, we assume that the
4
positions of all IRIF are equally distributed within the nucleus and that no two IRIF are closer
5
than 2 rg + 2 rp, which guarantees that a specific PML body does not colocalize with more than
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one IRIF at a time. Then, considering the volume of the nucleus and the total volume of the
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spheres around all IRIF which define a colocalization event, the colocalization probability p for
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one PML body is given by p = n ( rg + rp )3 / R3 . As for m PML bodies, the probability pm that no
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PML body colocalizes with any IRIF, is pm = (1-p)m , we get the final result for the probability
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pA to find at least one colocalization between a PML body and an IRIF within the whole nucleus
11
with m PML bodies and n genomic loci: pA = 1 - ( 1 - n ( rg + rp )3 / R3 )m. This model contains
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the explicit assumption that two IRIF have a minimum distance of 2(rp + rg), which is a plausible
13
assumption regarding the size of IRIF and of PML bodies. On the contrary, PML bodies are
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allowed to intersect in this probability model, which does not coincide with our experimental
15
findings. Nevertheless, for the numbers of PML bodies in our experiments, the effect of this
16
approximation on the true probability is very small. From the colocalizing probability pA within
17
one nucleus, one can compute the probability pk(j) for the event to find amongst k investigated
18
cells j cells with at least one colocalization caused by chance: pk(j) = (k \choose j) pAj (1 – pAj) (k-
19
j)
20
value for this event is the sum of the probabilities pk(j) to find q cells or more with at least one
21
colocalization: p-value = SUM(q≤i≤k) pk(i)
. If in an experiment with k cells there are q cells with at least one colocalization, then the p-
22 23
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1
Results
2 3 4
PML bodies are non-randomly associated with transient and persistent DNA damage foci In order to obtain a quantitative assessment of the association between irradiation-
5
induced foci (IRIF) and PML bodies, we analyzed their localization in WI-38 fibroblasts at
6
several time points after low (2 Gy) or high (15 Gy) dose γ-IR. Irradiation with 2 Gy and 15 Gy
7
induced a dose-dependent DNA damage response as detected by increased levels of p53 and p21,
8
but only the 15 Gy treatment lead to induction of cellular senescence as judged by increased
9
levels of p16 and SA-β-Gal (Fig. 1, A-C). Interstingly, there was a decreased expression of p21
10
(but not p53) 3 days after 2 Gy irradiation and a decreased expression of p53 and p16 (but not
11
p21) 3 days after 15 Gy irradiation (Fig, 1, A). We observed these expression patterns in several
12
independent experiments using Wi-38 fibroblastts and therefore exclude experimental artefacts.
13
These results suggest a 2-wave activation pattern for at least some cell cycle-related factors after
14
irradiation. Growth curves and phospho-pRB levels revealed that irradiation with 2 Gy induced a
15
transient cell cycle arrest with proliferation resuming 4 days after irradiation, while 15 Gy
16
irradiated cells displayed a senescence-associated permanent cell cycle arrest (Fig. 1, A and D).
17
Irradiation did not induce cell death (Fig. 1, E). As expected, increased numbers of IRIF were
18
detected 30 min after irradiation (Fig. 2, A and B). While the number of IRIF decreased to
19
control levels after 24 hrs in 2 Gy irradiated cells, nuclei of 15 Gy irradiated cells contained 10
20
(± 3) persistent DNA damage foci 6 d after irradiation (Fig. 2B).
21
Co-immunostaining of endogenous PML protein revealed that only a very small number
22
of IRIF are associated with PML bodies during the first 24 hrs after 2 Gy irradiation (Fig. 2A,
23
C). At later time points, the number of IRIF/PML body associations was as low as in control
10
1
cells (Fig. 2C). Association between PML bodies and IRIF was pronounced in 15 Gy irradiated
2
cells (Fig. 2A, inset). The number of IRIF/PML body associations was high at all time points
3
after irradiation with 15 Gy (Fig. 2C). The association rate peaks one day after irradiation (6 ± 1)
4
and declines to 3.5 (± 1.2) after 6 days. Qualitatively, we observed only a few association events
5
per nucleus irrespective of the time point of analysis and the damage load. Quantitation of
6
association events per nucleus revealed that not more than 30 % of IRIF were associated with a
7
PML body at all time points and irrespective of the damage load (Fig. 2D).
8
Since we also observed an irradiation-induced increase in the number of PML bodies
9
(Fig. 2E) it could not be fully excluded that an IRIF/PML body association occurs randomly in
10
the nucleus. We therefore employed a self-developed algoritm (48) to calculate the theoretical
11
probability of association events between IRIF and PML bodies. This required determination of
12
the number and volume of PML bodies and IRIF, and the nuclear volume at several time points
13
after irradiation (data not shown). With these data we determined that the theoretical probability
14
for only one random association between IRIF and PML bodies is extremely low (Fig. 2F). Thus
15
the topological association between PML bodies and IRIF is highly non-random at all time
16
points for up to one week during a γ-irradiation-induced DNA damage response in human
17
fibroblasts. Because the frequency of PML body/IRIF contacts in non-irradiated fibroblasts was
18
at least ten-fold higher than the theoretical value for such contacts we conclude PML bodies also
19
assemble at persistent (irreparable) DNA damage foci in fibroblasts without experimental
20
genotoxic stress.
21 22 23
To further analyze the spatial relationship between PML bodies and transient or persistent DNA damage foci we applied different doses of an UV-A laser microbeam to induce DNA
11
1
damage at 2-3 distinct loci in single nuclei. After irradiation with 2 µJ per pulse, Wi-38 cells
2
displayed DNA damage foci all of which disappeared 24 hrs after irradiation, most likely after
3
successful DNA damage repair (Fig. 3, A-D and data not shown). These transient DNA damage
4
foci were found to be associated with PML bodies 3 h after irradiation, corroborating that PML
5
bodies can specifically associate with transient DNA damage foci (Fig. 3C, yellow arrows). One
6
day after beam irradiation with 2 µJ per pulse the γH2AX signals had disappeared almost
7
completely. Only application of extreme artificial contrast stretching revealed residual γH2AX
8
dots, which did not colocalize with PML bodies (Fig. 3, enlargements with an asterisk).
9
Microbeam irradiation with 4 µJ or 8µJ per pulse produced DNA damage foci associated with
10
PML bodies as early as 30 min after irradiation (Fig. 3, E and I, yellow arrows). This indicates
11
that the timing of PML body assembly at IRIF is a function of the amount of damage within an
12
IRIF. An UV-A dose of 4 uJ or more induced persistent IRIF, which were not resolved after 24
13
hrs in Wi-38 fibroblasts. These unrepairable IRIF were associated with one or more PML body at
14
all time points of observation (Fig. 3, E-L).
15 16
Next we analyzed the spatial relationship between PML bodies and persistent DNA
17
damage foci in replicative senescent human and mouse primary fibroblasts. As expected, Wi-38
18
fibroblasts showed upregulation of the senescence markers p16, p21, p53, and SA-βgal during
19
serial passaging (Fig. 4A). Sixteen (± 4) % of γH2AX-foci in proliferating fibroblasts (PD 42)
20
were associated with PML bodies (Fig. 4B, yellow arrow; Fig. 4H). Replicative senescent Wi-38
21
fibroblasts at PD 58 accumulated 14 (± 3) persistent SDF per nucleus, of which 35% were found
22
to be associated with a PML body (Fig. 4, C and H). Freshly isolated wildtype MEFs contained
23
very low numbers of γH2AX foci (Fig. 4D). Senescence was induced using γ-irradiation, a
12
1
combination of the DNA-damaging drugs Bromodeoxyuridine and Distamycin A (BD), or by
2
serial passaging (Fig. 4, E-G). These treatments induced a 4 to 5 fold upregulation of the number
3
of DNA damage foci and 30% to 40 % of these foci were associated with a PML body (Fig. 4H).
4
These data show that PML bodies specifically assemble at DNA damage foci in senescent
5
primary human and mouse fibroblasts. However, the majority of these foci are not in contact
6
with a PML body (Fig. 4, C and E-G, red arrowheads, and H). In order to assess if PML
7
body/damage foci contacts are random or specific, we compared the number of actually observed
8
contacts with the theoretical probability for random associations as determined with a computer
9
program (48, see Materials & Methods) (Table 1). This analysis showed for example that the
10
probability for only one random contact between a PML body and a peristent DNA damage site
11
is ∼7 %. This means that in 7 out of 100 cells one contact can occur randomly. However, we
12
measured on average 5 such contacts in each and every cell nucleus. Similarly, the probability of
13
the randomness of the observed association rates in irradiated cells at different time points after
14
irradation was always always below 1%, indicating a specificity of interaction of at least 2 orders
15
of magnitude higher than random association events (Table 1).
16
Previously it was reported that irradiation-induced DNA damage foci in mouse lung cells
17
are associated with PML bodies (19). We sought to extend these observations by analyzing non-
18
irradiated tissue. Immunofluorescence analyses of thin sections of human skin samples revealed
19
a high number of PML body/pATM foci associations (Fig. 5, A and B). PML bodies were found
20
to be associated with 30 (± 18) % and 47 (± 12) % of pATM–containing DNA damage foci in
21
cells of the epidermis and dermis, respectively (Fig. 5C). These observations demonstrate that a
22
close physical relationship between PML bodies and DNA damage foci does not only occur after
23
irradiation, but may also be a hallmark of unperturbed human tissue. PML body/DNA damage
13
1
foci association was also observed in human skin tissue when anti-γH2AX antibodies were used
2
(Fig. 5D). pATM foci in cell nuclei of tissues are indicative of persistent DNA damage foci in
3
senescent cells (16). Senescent cells in human skin tissue are characterized by the accumulation
4
of annexin A5 (ANX5) in the periphery of the cell nucleus (45). When ANX5 and pATM were
5
immunostained simultanuously in epidermal skin layers, we observed that the majority of pATM
6
foci-poistive cells also expressed ANX5, indicating that these are senescent cells (Fig. 5, E and
7
F). This provides strong support for our conclusion that PML bodies are associated with
8
persistent DNA damage foci in human skin. Taken together, our data demonstrate a highly non-
9
random association between PML bodies and IRIF or senescence-associated DNA damage foci
10
in various cell culture systems, as well as in normal human skin.
11 12 13
DNA damage response factors do not accumulate in PML bodies during senescence PML bodies attached to SDFs may contribute to sustained DNA damage signaling in
14
senescent cells. In order to address this hypothesis we analyzed if DNA damage response (DDR)
15
factors become transiently associated with PML bodies during a DNA damage response. Control
16
experiments confirmed that in Wi-38 fibroblasts all PML bodies also contained Sp100 during a
17
DNA damage response, in DNA damage-induced senescent cells and in replicative senescent
18
fibroblasts (data not shown). Therefore, anti-Sp100 antibody staining is an appropriate means to
19
detect PM bodies in co-innumo staining analyses. MDC1 accumulated at γH2AX foci 30 minutes
20
after irradiation with 2 Gy (Fig. 6B). One day after irradiation, γH2AX foci had disappeared and
21
MDC1 resumed its diffuse nucleoplasmic distribution (Fig. 6C). Importantly, at no time point
22
after irradiation with 2 Gy or 15 Gy did we observe any colocalization of MDC1 with PML
23
bodies (Fig. 6 B-E, insets). Likewise, neither 53BP1 nor phosphorylated NBS1 were found to
14
1
colocalize with PML bodies adjacent to unrepaired DNA damage foci (Fig. 6, D-G). PML bodies
2
appeared to be excluded from γH2AX-labeled chromatin, while the DDR factors completely
3
colocalized with the γH2AX signal (Fig. 6, D-G, insets). The same result was obtained in
4
replicative as well as DNA damage-induced senescent fibroblasts (Fig. 6, H and I). In
5
irradiation-induced senescent Wi-38 fibroblasts, p16, p21 and p53 were found diffusely
6
distributed in a micro-punctate pattern throughout the nucleus (Fig. 6, J-L). Although some of
7
the microdots spatially coincided with the position of a PML body in confocal images (Fig. 6, J-
8
L, linescans) there was no substantial enrichment of p16, p21 or p53 within the center of PML
9
bodies (Fig. 6, J-L, insets). Our data therefore indicate that the DDR factors MDC1, 53BP1,
10
phospho-NBS1, p16, p21 and p53 are not components of PML bodies during a DNA damage
11
response or in senescent cells.
12 13
During DNA repair, all PML bodies are in contact with RPA/Rad51 containing repair foci
14
To analyze the association between PML bodies and sites of DNA damage during ongoing repair
15
in more detail we applied 3-dimensional structured illumination microscopy (3D-SIM). SIM
16
provides an opitcal resolution limit of ∼100 nm. Human fibroblasts were irradiated with 10 Gy
17
and immunostained 3 hrs later to detect PML bodies, γH2AX foci and RPA (Fig. 7, A). At that
18
time point cell nuclei contained 25 (± 7) PML bodies and 68 (± 17) γH2AX foci (mean ± SD, n =
19
20 nuclei). The super-resolution images revealed that 91 (± 7) % of all γH2AX foci in a cell
20
nucleus (n = 10) overlapped with at least one RPA focus (i.e. Fig. 7, A-1 and A-2). Twenty-two
21
(± 5) % of the latter were also in contact with a PML body (i.e. Fig. 7, A-1). Interestingly,
22
γH2AX foci without an associated RPA focus were never found in contact with a PML body,
23
while all RPA foci without an associated γH2AX focus were always in contact with a PML
15
1
body. In other words: During ongoing DNA repair (3 hrs after 10 Gy) all PML bodies are in
2
contact with either a γH2AX or a RPA focus or with both at the same time. Very similar
3
observations were made when Rad51 foci were analyzed in combination with γH2AX foci and
4
PML bodies by SIM (Fig. 4, J).
5 6 7
PML depletion does not substantially impair the DNA damage response. As PML NBs associate with DNA damage foci, the question arises, whether cells lacking
8
PML show an impaired DNA damage response. To this end we used primary MEFs freshly
9
isolated from PML wildtype, heterozygous or knockout embryos, as well as primary human
10
foreskin fibroblasts stably expressing an shRNA targeting all nuclear PML isoforms. These cells
11
did not express detectable amounts of PML protein or PML nuclear bodies (48, and data not
12
shown). Western blot analyses of irradiated MEFs revealed no differences of the irradiation-
13
induced phosphorylation of H2AX, KAP1 or 53BP1 when cells with or without endogenous
14
PML were compared (Fig. 8A). Measurement of the protein levels from several independent
15
Western blots also showed no statistically significant differences in the activation levels of these
16
DDR factors in PML proficient vs. deficient MEFs after irradiation (Fig. 8, B and C). We then
17
analyzed DNA damage foci formation in PML-knockout MEFs. Irradiation with 2 Gy induced
18
formation of numerous γH2AX foci after 30 min and these foci completely colocalized with
19
53BP1 immunostaining (Fig. 9, A). DNA damage foci formation in MEFs was quantified over
20
time after irradiation with 2 or 15 Gy. The results indicated no significant difference in the
21
efficiency of foci formation (Fig. 9, B and C). These observations show that PML does not play
22
an important role in IRIF formation and the DNA damage response in primary MEFs. In
23
contrast, PML-depleted human fibroblasts showed a significantly reduced number of DNA
16
1
damage foci 30 min and 3 hrs after 2 Gy irradiation but not at later time points (Fig. 9, D).
2
However, IRIF formation was not changed in PML-depleted human fibroblasts after 15 Gy
3
irradiation (Fig. 9, E). These observations suggest a functional role for PML in IRIF maintenance
4
during the DSB repair process after low dose but not high dose irradiation in human fibroblasts.
5 6 7
Lack of PML does not alter DNA damage-induced or replicative senescence Next, we addressed the question whether DNA damage-induced senescence is impaired
8
in the absence of PML. Control and PML-depleted human fibroblasts were irradiated with 15
9
Gy. As shown by SA-β-Gal-staining, the lack of PML has no significant effect on the efficiency
10
of senescence induction after 6 days (Fig. 10, A). A similar result was obtained when
11
BrdU/DMA was used to induce senescence (Fig. 10, B). BrdU/DMA treatment also induced
12
persistent DNA damage foci in freshly isolated wildtype MEFs but their number is not altered in
13
the absence of PML (data not shown). Notably, the efficiency of BrdU/DMA-induced
14
senescence is not changed in PML-/- MEFs (Fig. 10, C). Similarly, there is no difference between
15
wt and PML-/- MEFs after radiation-induced senescence (Fig. 10, C). These data demonstrate,
16
that the PML protein is dispensable for DNA damage-induced senescence in primary human and
17
murine fibroblasts. Finally we analyzed the replicative potential of human and mouse fibroblasts
18
expressing or lacking PML. Primary human fibroblasts ceased proliferation after 90 to 100 days
19
of serial passaging (Fig. 10, D). Surprisingly, PML depleted fibroblasts reached PD38, while
20
PML containing cells yielded about 10 population doublings more. This result suggests that a
21
stable knockdown of PML in primary human fibroblasts acts anti-proliferative. We observed this
22
phenomenon in several independent stable human PML knockdown cell lines (data not shown).
23
In contrast, MEFs entered the replicative senescence-induced proliferation arrest after 10 to 12
17
1
days irrespective of the endogenous PML expression level (Fig, 10, E). These observations
2
suggest that PML does not play a major functional role in the course of replicative senescence, at
3
least in MEFs.
4 5 6
Discussion
7
Numerous models exist for the role of PML protein or the nuclear bodies in the DDR. It was
8
suggested that NBs may serve as DNA repair compartments (27), as DNA damage sensors (28,
9
30), or as a modification platform for DNA damage-activated p53 and other DDR factors (23,
10
39, 42, 43, 49, 50). Telomeric and non-telomeric DNA damage efficiently initiates and maintains
11
senescence (9). Since PML has been functionally implicated in senescence induction (21, 22, 33,
12
40), we expected to find a functional link between PML and DNA repair and DNA damage
13
induced senescence. Surprisingly, our data demonstrate that this is not the case for human and
14
murine primary fibroblasts.
15 16 17
PML bodies associate with persistent DNA damage foci in vitro and in vivo We confirmed that PML NBs non-randomly associate with persistent DNA damage foci
18
after γ-IR, UV-A-IR, or radio-mimetic drugs (17, 19, 30). In extension of such analyses we
19
observed that an association between PML bodies and IRIF only occurs at late time points after
20
damage induction by γ-IR or after high-dose and spatially concentrated UV-A irradiation. In
21
contrast, irradiation with low doses of γ-IR (≤ 2 Gy), which inflicts repairable DNA damage in
22
primary fibroblasts (Fig. 1A, B) did not increase the association rate between PML bodies and
23
IRIF. We therefore conclude that PML bodies do not act as immediate early sensors of DNA
18
1
damage. This assumption is corroborated by our observation that 2 µJ UV-A microbeam
2
irradiation induces PML body/IRIF associations only 3 hrs after the irradiation pulse (Fig. 3). On
3
the other hand, higher doses of UV-A induced PML/IRIF associations already after 30 min,
4
consistent with previous observations (30). The timing of PML recruitment may therefore be a
5
function of the number of DSBs in an IRIF (28).
6
Notably, only about 30% or 50% of persistent DNA damage foci are associated with a
7
PML body in vitro or in vivo, respectively. In HCA2 human foreskin fibroblasts the frequency of
8
association appears to be higher (19). Nevertheless, although the number of contacts may differ
9
between cell types, nuclei always contain DNA damage foci which are not in contact with a
10
PML body. Dellaire and colleagues could show with the help of electron microscopy, that the
11
structure of these persistent foci does not depend on the spatial proximity to a PML NB (16).
12
Thus, PML bodies are dispensable for the maintenance of persistent DNA damage foci.
13
Persistent DNA damage has been proposed to be an important trigger for senescence
14
induction (4, 6, 51, 52). This suggested that an association of PML with persistent foci is a
15
marker for pre-senescent and senescent cells (19). Here we confirmed this assumption for human
16
skin. These foci, also referred to as senescence-derived foci (SDFs) (4, 9, 53), may represent
17
clustered DNA damage sites (54, 55), difficult-to-repair DNA damage in heterochromatin (56) or
18
uncapped telomeres (4, 5, 57). DNA synthesis was not detected in persistent foci (19). But, as
19
they still recruit DNA damage factors, it is very likely that they transmit a DNA damage signal,
20
which is sufficient to maintain a DDR and also to establish a cell cycle arrest and senescence (51,
21
52). This is consistent with the observation that self-organized assembly of canonical DDR
22
factors on chromatin in the absence of DNA damage is sufficient to induce a DDR and cell cycle
23
arrest (51). Since DNA damage-induced senescence is not compromised in fibroblasts lacking
19
1
PML (Fig. 10) we conclude that PML nuclear bodies are dispensable for senescence-maintaining
2
DDR signaling from IRIF or persistent foci. Although the PML body associated pATM foci
3
observed here in human skin tissue fulfilled all criteria which define persistent DNA damage foci
4
(size, morhopology, ANX5-positive senescent cells, association with PML bodies) (11, 13, Fig.
5
5E,F), we cannot fully exclude the possibilty that these pATM foci may be of transient nature.
6 7 8 9
Potential function of PML at IRIF The present study confirmed that PML bodies associate with irradiation-induced as well
10
as endogenous persistent DNA damage foci (17, 19). These observations suggested a model in
11
which PML bodies form at sites of persistent DNA damage from where DNA damage response
12
signalling may be regulated (17, 18). The model implied that foci formation may be impaired in
13
the absence of PML. Analyses in the present report revealed that the formation of DNA damage
14
foci is not impaired in PML-deficient human or mouse primary fibroblasts after senescence-
15
inducing doses of γ-irradiation. However, human primary fibroblasts lacking PML show
16
significantly reduced numbers of IRIF after low dose irradiation (2 Gy) (Fig. 9, D). This is
17
consistent with recently published studies showing that accumulation of many DDR factors at
18
DNA damage foci is abolished in PML-depleted cancer cells (18, 58-60). In primary human
19
embryo lung fibroblasts, the clearance of γH2AX foci after 10 Gy of irradiation was shown to be
20
reduced in siPML treated cells (60). Interstingly, the formation of IRIF was not impaired in this
21
setting since the number of γH2AX foci after 30 min of irradiation was not changed (60). In
22
contrast, at 2 Gy we observed here a significantly reduced number of IRIF in PML-depleted
23
human fibroblasts after 30 min of irradiation (Fig. 9D). Collectively these observations suggest
20
1
that PML (bodies) may contribute to both formation and clearance of IRIF. In contrast to this
2
conclusion, depletion of PML in HT 1885 fibrosarcoma cells has no impact on γH2AX levels
3
and formation of Rad51 foci (59). The discrepancy to our and previous (60) results may be
4
explained by differences in the experimental set-up, mainly the usage of a cancer cell line in the
5
study by Yeung et al. (59). Immortal cells may have compromised DNA damage response
6
activities as compared to primary cells used in our study. Overmore we consider our analysis of
7
counting individual IRIF at the single cell level more sensitive than determination of γH2AX
8
fluorescence over a population of cells. In contrast to human cells, IRIF formation after 2 Gy was
9
not impaired in PML knockout MEFs (Fig. 9, B) indicating cell or species-specific roles for
10
PML in the DDR.
11
Homology-directed repair (HR) on artificial reporter plasmids is impaired in PML-depleted
12
BJ/tert and HT1885 cells (58, 59). PML bodies normally do not contain DNA (16, 61), but
13
accumulate factors involved in DNA repair, especially in homologous recombination (HR), e.g.
14
BLM (26, 62-64), WRN (60, 65), RPA (19, 26, 30, 66) and BRCA1 (18). As this pathway
15
involves extensive formation of single-stranded DNA (67), PML bodies may help to organize
16
single-stranded chromatin structure during HR. This is consistent with the observation that (i)
17
PML bodieas are associated with ssDNA foci, (ii) that formation of these foci is impaired after
18
PML-depletion, and that PML ko MEFs have an elevated rate of sister chromatin exchange (27;
19
64). We have shown here for the first time by superresolution micrsocopy, that during ongoing
20
irradiation-induced repair virtually all PML bodies are engaged at γH2AX, Rad 51, or RPA foci,
21
or combinations of them (Fig. 7). It had been shown previously for human fibroblasts that both,
22
Rad51 and RPA specifically accumulate at irradiation-induced ssDNA-containing nuclear foci
23
(68). Together, these observation strongly implicate a function for PML nuclear bodies in the
21
1
formation and/or maintenance of ssDNA-containing repair foci. Since γH2AX foci without an
2
associated RPA or Rad51 focus were never found in contact with a PML body we suggest that
3
PML bodies assemble at γH2AX foci only when these contain ssDNA.
4
The super-resolution images also revealed that PML bodies, γH2AX foci, and RPA or
5
Rad51 foci overlapped with each other but never fully colocalized. This is in contrast to
6
published data showing accumulation at PML bodies of many DDR factors (18, 19, 26, 30, 62-
7
66). The discrapancy may be explained by the usage of different cell types in the variuos studies.
8
We believe that our observations are more conclusive because we used super-resolution
9
microscopy on primary cell types. In addition, many previous studies used cancer cell lines,
10
including alternative lengthening of telomeres (ALT)-positive lines. ALT is a DNA
11
recombination event that occurs at specialized PML bodies (APBs) at telomeres with telomaric
12
DNA inside the APBs. Finally, some of the previous studies used a pre-permeabilization step
13
before fixation of cells. It has been shown recently that such protocols can induce the collapse of
14
adjacent cellular complexes including nuclear substructures, potentially leading to fixation-
15
induced colocalization events (69).
16 17
PML bodies are non-essential in DNA damage-induced cellular senescence
18
There is strong evidence that PML is involved in cellular senescence based on over-
19
expression of PML isoform IV (PML 3) (32, 34, 35, 70, 71). PML knockout or knockdown
20
fibroblasts appear to be resistant to Ras-induced and TGFβ-induced senescence (32, 40, 42, 72).
21
In the present report we have investigated the role of PML in DNA damage-induced senescence
22
and found that in contrast to Ras- or TGF-β-induced senescence, the function of PML is not
23
essential for senescence induction after DNA damage in primary human and murine fibroblasts
22
1
(Fig. 10). In contrast, thymocytes from PML-/- mice are partially resistent to irradiation-induced
2
apoptosis (37) again suggesting cell type specific functions of PML.
3
We also could not detect an accumulation of the cell cycle inhibitors p53, p21 and p16 at
4
endogenous expression levels at PML bodies (Fig. 6). This was surprising, as other groups have
5
observed stress-induced colocalization of PML and p53 (17, 32, 42, 50, 73). Finally, at least
6
PML-depleted primary MEFs are not resistant to or show accelerated replicative senescence
7
(Fig. 10 E). In contrast, primary human fibroblasts lacking PML displayed a somewhat reduced
8
replicative potential as they did not reach the maximum number of population doublings
9
observed in control fibroblasts (Fig. 10 D). This was unexpected since PML is believed to have
10
tumor supressor activities. We have no ready expnation for this phenomenon and further studies
11
are required to unravel the mechanisms of the stable PML depletion induced proliferation
12
inhibition during long-term passaging of human fibroblasts.
13 14
In conclusion, our data show that the association of PML NBs with persistent DNA
15
damage foci is characteristic of senescent cells in cell culture and in vivo. In contrast to Ras- and
16
TGF-β-induced senescence, the function of PML NBs is not essential for senescence induction
17
after DNA damage in cells from both species. However, we would like to stress that PML
18
association at persistent DNA damage sites should still be considered to be a cellular signal to
19
promote apoptosis and senescence, PML probably is just one of several partly redundant factors
20
that may provide this signal. Our findings extend and refine our understanding of the role of
21
PML NBs in tumor suppression. It becomes apparent that more data are needed on the effect of
22
different stressors and senescence inducers in analyses of PML functions. Besides, a careful
23
1
discrimination between the signal pathways in different cell types and species is necessary, when
2
PML is investigated with respect to stress-induced cellular responses.
3 4
Acknowledgements
5
We thank S. Ohndorf and M. Koch for technical assistance. We are greatful to Hans Will and
6
Hannah Stäge (HPI, Hamburg, Germany) for providing PML knockout mice and help in
7
isolation of MEFs, Nina Reuter and Thomas Stamminger (University of Erlangen, Germany) for
8
providing stable PML knockdown fibroblasts and PML-specific shRNA plasmids, Johannes
9
Norgauer and Mirjana Zimmer (University hospital Jena, Germany) for providing skin tissue;
10
Anja Krüger and Tjard Jörß (animal facility, FLI, Jena, Germany) for patient help and support in
11
work with mice and preparation of primary cells. We would also like to thank Stephan Diekmann
12
and Tobias Ulbricht for enlightning discussions and lab support. This work was supported by
13
grant HE 2484/3-1 from the Deutsche Forschungsgemeinschaft. We also acknowledge JenAge
14
funding by the German Ministry for Education and Research (Bundesministerium für Bildung
15
und Forschung – BMBF; support code: 0315581).
16 17 18 19 20
Figure legends
21
Figure 1. Irradiation-induced senescence in WI-38 fibroblasts.
22
(A) WI-38 fibroblasts were γ-irradiated with 2 Gy or 15 Gy and whole cell lysates were analyzed
23
by immunoblotting to detect the indicated proteins. (B) Representative images of SA-βgal
24
1
staining of WI-38 cells 6 d after γ-IR. Quantification of SA-βgal staining (C), number of living
2
cells (D) and number of dead cells (E) within 6 days after irradiation. At least 50-100 cells per
3
time point and irradiation dose were monitored. All experiments were done in triplicates. Mean
4
values ± SEM are depicted.
5
Figure 2. PML NBs non-randomly associate with persistent DNA damage foci.
6
(A) Indirect immunofluorescence staining of WI-38 fibroblasts after γ-IR. Unirradiated (0 Gy)
7
and irradiated cells (2 Gy or 15 Gy) were fixed at indicated time points and stained with
8
antibodies against PML and γH2AX. Scale bar 5 µm. (B-F) Quantification of γH2AX foci (B),
9
association events between PML NBs and γH2AX (C), percentage of γH2AX-foci associated
10
with PML NBs (D), number of PML NBs (E) and theoretical probability for one random
11
association between PML NBs and γH2AX foci (F). Quantification of foci and potential
12
associations were carried out using the Image J software and customized macros. Mean values ±
13
SEM are depicted.
14
Figure 3. PML NBs associate with UV-A microirradiation-induced foci.
15
WI-38 fibroblasts were irradiated with different doses of an UV-A microbeam, fixed at indicated
16
time points and immuno-stained to detect PML (green) and γH2AX (red). Regions within nuclei
17
marked by a white box are shown as magnified views (seperated into single color channels
18
shown as grey scale images) on the right side of each overview panel. Note that in the panels
19
marked by an asterisk (in D) the signals for γH2AX were contrast-streched to visualize residual
20
fluorescence. Scale bar = 5 µm
25
1
Figure 4. PML NBs associate with DNA damage foci in senescent human and murine cells.
2
(A) WI-38 fibroblasts at different population doublings (PD) were subjected to Western blot
3
analysis using antibodies against the indicated proteins (top panels). The number of SA-βgal
4
positive cells during serial passaging of these cells was also monitored (bottom graph).
5
Representative mid-nucleus confocal images of young (B), replicative senescent (C),
6
unirradiated primary MEFs (D), 15 Gy-irradiated MEFs after 6 d (E), MEFs treated with BrdU
7
and DMA for 6 d (F) and replicative senescent MEFs (passage 6, PD 12) (G). Cells were fixed
8
and immuno-stained to detect PML (green) and γH2AX (red). Percentage of SA-βgal-positive
9
cells is indicated (mean ± SD). (H) Quantification of γH2AX-foci (grey bars) and percentage of
10
γH2AX-foci associated with a PML NBs (blue bars) for the indicated cells. Freshly, isolated
11
primary MEFs (passage 1) were used for irradiation and drug treatment, replicative senescent
12
MEFs were at passage 6 (PD 12). Experiments were done in triplicates. Mean values ± SEM are
13
depicted. (I, J)
14 15
Figure 5. PML NBs associate with DNA damage foci in human skin cells.
16
Cryosections of human skin were stained with antibodies against PML (green) and
17
phosphorylated ATM (red). Representative confocal images of the epidermis (A) and the dermis
18
(B) are shown. The percentage of pATM-positive cells as well as the association between pATM
19
foci and PML NBs was quantified in five skin sections from different donors (C). Mean values ±
20
SEM are depicted. (D) Antibodies against γH2AX (red) also revealed contacts between DNA
21
damage sites and PML bodies (green) in the dermis of human skin. (E) Confocal image of a
22
human skin section immunostained with antibodies against annexin A5 (ANX, green) and pATM
23
(red). DNA was stained with DAPI. (F) Quantitation of the number of cells positive for the
26
1
indicated events from experiments shown in (E). Mean values ± SEM are depicted (n = 10
2
individual healthy skin sections from 3 different, mid-aged donors).
3
Figure 6. MDC1, 53BP1, pNBS1, p16, p21 and p53 do not accumulate at persistent DNA
4
damage-associated PML NBs.
5
WI-38 fibroblasts were γ-irradiated and fixed at indicated time points after DNA damage
6
induction (A-H) or passaged until senescence (I). Fixed cells were subjected to
7
immunofluorescence staining. An antibody against Sp100 was used as a marker for PML NBs
8
combined with a γH2AX antibody to stain DNA damage foci. Additionally, the DNA damage
9
response proteins Mdc1, 53BP1 and phosphorylated Nbs1 were stained. (J-L) WI-38 fibroblasts
10
were γ-irradiated with 15 Gy, fixed after 6 days and stained with antibodies against p16, p21 or
11
p53 and co-stained with an antibody against PML. Line scans (panels on the right) show the pixel
12
intensity distribution along the lines indicated in the merge image. Scale bar = 5 µm.
13
Figure 7. Super resolution imaging of PML nuclear bodies and DNA damage sites. Primary
14
human fibroblasts (at low PD) were irradiated with 10 Gy, fixed after 90 min and subsequently
15
immunostained to detect the indicated proteins. 3D-SIM super-resolution images of single nuclei
16
were collected using an ELYRA structured illumination microscope (Zeiss). White boxes within
17
the overview images are shown enlarged in the right hand panels. Each right hand panel in
18
addition shows the individual channels in monochrome.
19
Figure 8. DNA damage signaling is not altered in PML knockout MEFs.
20
(A) PML wildtype, heterozygous and knockout MEFs were γ-irradiated, lysed at indicated time
21
points and Western blot analyses were carried out using the indicated antibodies. (B, C) Relative
22
protein levels as measured by densitometry of Western blot bands were quantified using ImageJ
27
1
software. All experiments were done in triplicates. Mean values ± SEM are depicted. Note that
2
the panels shown in (A) have been spliced from two Western blots each. The splice sites are
3
indicated by a thin white line between 3 and 8 hrs or 0.5 and 3 hrs within the left and the right
4
panel, respectively. All Western blots shown in (A) originate from the same gel run and were
5
developed simultanously under the very same conditions, which also allows comparison of the
6
relative band intensities even between the 2 Gy and 15 Gy irradiated cells.
7 8
Figure 9. IRIF formation in PML-depleted cells.
9
(A) Freshly isolated MEFs from PML wildtype, heterozygous and knockout embryos were
10
irradiated with 2 Gy and immunolabeled 30 min later using antibodies against γH2AX (green)
11
and 53BP1 (green). Images show representative mid-nucleaus confocal sections. Bars = 5 µm.
12
(B, C) MEFs as in (A) were irradiated with 2 Gy or 15 Gy and samples on coverslips were taken
13
at different time points after irradiation. Cells were immunolabeled as in (A) to quantify the
14
number of DNA damage foci Data points represent mean values ± SEM. (D, E) Same is in (B, C)
15
using primary human fibroblasts stably expressing a vector control, vector encoding control
16
siRNA and vector expressing siRNA against PML. Data points represent mean values ± SEM.
17 18
Figure 10. PML depletion does not alter fibroblast senescence.
19
Primary human fibroblasts with a stable PML-knockdown (siPML) or control-infected cells
20
(vector, siControl) were (A) γ-irradiated with 15 Gy and fixed after 6 days or (B) treated with
21
with 50 µM BrdU and 10 µM DMA and fixed at indicated time points. To monitor senescence
22
induction, cells were stained for SA-βgal. (C) pMEFs isolated from PML wildtype, heterozygous
28
1
and knockout embryos were treated with with 50 µM BrdU and 10 µM DMA, or 20 Gy
2
irradiation, fixed after 6 days and stained for SA-βgal activity. In all experiments at least 50 cells
3
were monitored and all experiments were done in triplicates. Mean values ± SEM are depicted.
4
HFFs with a stable PML-knockdown (siPML) or control-infected cells (vector, siControl) (D) as
5
well as wild-tyoe (+/+). heterozygous PML knockout (+/-), and PML knockout (-/-) pMEFs (E)
6
were cultured, until they reached senescence and population doublings over time were
7
monitored. For pMEFs 2-3 cell lines per genotype were used and mean values ± SEM are
8
depicted.
9 10
Table 1: The association between PML bodies and γH2AX foci is highly non-random
11
The average number (n = 100 nuclei each) of γH2AX foci and of PML bodies and their
12
association rate were determined in young Wi-38 fibroblasts after irradiation and in replicative
13
senescent cells as indicated. The probability of these association was determined using a self-
14
developed computer algorithm (Ref 48; Materials & Methods)
15 16 17
29
1
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38
1
Figure 1
2 3
39
1
Figure 2
2
40
1
Figure 3
2
41
1
Figure 4
2 3
42
1
Figure 5
2
43
1
Figure 6
2
44
1
Figure 7
2
45
1
Figure 8
2
46
1
Figure 9
2
47
1
Figure 10
2 3 4
48
1 time after irradiation
2 Gy
15 Gy
replicative senescence
number of number of gH2AX foci PML NBs
number of associations per nucleus
probability of observed association rate being random in %
probability of 1 random association in %
0
5
10
1
0,14
0,14
0.5 h
29
8
2
0,01
1,42
1d
6
10
1
0,93
0,93
3d
6
11
1
0,82
0,82
6d
4
13
1
0,22
0,22
0
5
10
1
0,14
0,14
0.5 h
62
10
5
0
1,09
1d
27
13
6
0
3,92
3d
8
14
3
0
1,12
6d
10
24
3
0
5,43
-
13
25
5
0
7,28
!
2 3
Table 1: The association between PML bodies and γH2AX foci is highly non-random
4
The average number (n = 100 nuclei each) of γH2AX foci and of PML bodies and their
5
association rate were determined in young Wi-38 fibroblasts after irradiation and in replicative
6
senescent cells as indicated. The probability of these association was determined using a self-
7
developed computer algorithm (Ref 48; Materials & Methods)
49
