MOLECULAR AND CELLULAR BIOLOGY, Jan. 2009, p. 68–82
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 29, No. 1
Death Receptor-Induced Activation of the Chk2- and Histone
H2AX-Associated DNA Damage Response Pathways?
Ste ´phanie Solier, Olivier Sordet, Kurt W. Kohn, and Yves Pommier*
Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892-4255
Received 9 April 2008/Returned for modification 5 June 2008/Accepted 21 October 2008
TRAIL is an endogenous death receptor ligand also used therapeutically because of its selective proapoptotic
activity in cancer cells. In the present study, we examined chromatin alterations induced by TRAIL and show
that TRAIL induces a rapid activation of DNA damage response (DDR) pathways with histone H2AX, Chk2,
ATM, and DNA-PK phosphorylations. Within 1 h of TRAIL exposure, immunofluorescence confocal micros-
copy revealed ?-H2AX peripheral nuclear staining (?-H2AX ring) colocalizing with phosphorylated/activated
Chk2, ATM, and DNA-PK inside heterochromatin regions. The marginal distribution of DDR proteins in early
apoptotic cells is remarkably different from the focal staining seen after DNA damage. TRAIL-induced DDR
was suppressed upon caspase inhibition or Bax inactivation, demonstrating that the DDR activated by TRAIL
is downstream from the mitochondrial death pathway. H2AX phosphorylation was dependent on DNA-PK,
while Chk2 phosphorylation was dependent on both ATM and DNA-PK. Downregulation of Chk2 decreased
TRAIL-induced cell detachment; delayed the activation of caspases 2, 3, 8, and 9; and reduced TRAIL-induced
cell killing. Together, our findings suggest that nuclear activation of Chk2 by TRAIL acts as a positive feedback
loop involving the mitochondrion-dependent activation of caspases, independently of p53.
Programmed cell death, apoptosis, is a normal physiological
process in which damaged or harmful cells are eliminated. It is
essential for tissue homeostasis, providing a balance between
survival and cellular destruction. Deregulation of apoptosis is a
common characteristic of many diseases, including cancers and
autoimmune disorders (66). Apoptosis consists of intrinsic and
extrinsic pathways. The intrinsic pathway can be initiated by
DNA or tubulin damage and engages the mitochondria. The
extrinsic pathway is initiated by members of the TNF (tumor
necrosis factor) superfamily and engages the death-inducing
signaling complex (DISC) at the cell surface (14).
TNF-related apoptosis-inducing ligand (TRAIL), a member of
the TNF/death receptor (DR) gene superfamily, is a physiological
endogenous ligand and a promising cancer therapy because it
induces apoptosis preferentially in cancer cells (29, 37). TRAIL
can bind to four plasma membrane receptors and one soluble
receptor, i.e., TRAIL-R1 (DR4), TRAIL-R2 (DR5/KILLER),
TRAIL-R3 (DcR1), TRAIL-R4 (DcR2), and osteoprotegerin,
respectively (26). DR4 and DR5 contain a common conserved
death domain motif. After binding TRAIL, DR4 and DR5 form
trimeric complexes and generate the DISC. In the DISC, the
adaptor protein FADD (Fas-associated death domain) binds to
the DR and to procaspase 8, promoting the autoactivation of this
caspase. DISC-activated caspase 8 engages the downstream
apoptotic death machinery in two ways. In type I cells, DISC
assembly activates sufficient amounts of caspase 8 to cleave and
activate downstream effector caspases. In type II cells, DISC as-
sembly activates smaller amounts of caspase 8 and requires am-
plification of the apoptotic signal through the mitochondrial
apoptotic pathway. Activation of this mitochondrial amplification
loop is achieved through cleavage of Bid, a proapoptotic member
of the Bcl-2 family (33). Cleaved Bid binds to and activates Bax
and Bak, which causes the release of apoptogenic factors such as
cytochrome c from mitochondria. In turn, cytochrome c activates
effector caspases via Apaf-1 and caspase 9 in the apoptosome
Preclinical evaluations demonstrated that recombinant TRAIL
inhibits tumor growth and induces the regression of a broad
range of leukemia and solid malignancies (26). Clinical trials with
TRAIL (www.gene.com/gene/pipeline/status/oncology/apo2l/) and
DR antibodies are ongoing (53). HGS-ETR1 (a human agonistic
monoclonal antibody that targets DR4) is in clinical trial in
combination with bortezomib against advanced multiple my-
eloma and in combination with carboplatin/paclitaxel and cis-
platin/gemcitabine against various advanced solid malignan-
cies. HGS-ETR2 (a human agonistic monoclonal antibody that
targets DR5) is in phase I trial with patients with advanced
solid malignancies. In contrast to monoclonal antibodies to
TRAIL receptors, TRAIL interacts with both DR4 and DR5,
as well as with the decoy receptors DcR1 and DcR2. Thus,
recombinant TRAIL may have a wider therapeutic spectrum
than the highly specific antibodies.
One of the biochemical landmarks of apoptosis is the for-
mation of DNA double-strand breaks (DSB; producing oligo-
nucleosomal DNA fragments and in some cells only large
fragments [50 to 300 kbp]) (55). Under conditions unrelated to
apoptosis, DSB induce the rapid activation of conserved DNA
damage response (DDR) pathways (56). Chk2 (checkpoint
kinase 2) becomes phosphorylated at threonine 68 (T68) by
several members of the phosphatidylinositol-3-kinase family,
i.e., ATM (ataxia telangiectasia mutated), ATR (ATM and
Rad3 homolog), and DNA-PK (DNA-dependent protein ki-
nase) (2, 31, 43). In addition to its cell cycle and checkpoint/
DNA repair functions, Chk2 has a proapoptotic function,
which is mediated in part by p53 (6, 20, 23, 25, 35, 58).
* Corresponding author. Mailing address: Bldg. 37, Rm. 5068, NIH,
Bethesda, MD 20892-4255. Phone: (301) 496-5944. Fax: (301) 402-
0752. E-mail: email@example.com.
?Published ahead of print on 27 October 2008.
One prominent chromatin modification in response to DSB
is phosphorylation of histone H2AX on serine 139, which is
referred to as ?-H2AX (48). Both ATM and DNA-PK catalyze
H2AX phosphorylation (5, 15, 38), and endogenous activation
of the DDR pathways has been observed in early tumorigen-
esis (3, 13). ?-H2AX has also been associated with apoptosis
(38, 50, 60). The focus of the present study was to determine
whether TRAIL induces the activation of DDR pathways and
whether this activation could have a functional impact on
apoptosis. We also describe and analyze a previously uniden-
tified marginal and confluent (ring) staining of ?-H2AX in
early apoptotic cells.
MATERIALS AND METHODS
Chemicals. Recombinant human soluble TRAIL was obtained from Alexis
Biochemicals (Axxora, San Diego, CA). The broad-spectrum caspase inhibitor
Z-VAD-fmk (z-Val-Ala-DL-Asp-fluoromethylketone) was from Bachem (Tor-
rance, CA). The DNA-PK kinase inhibitor (DNA-PKi; NU7441) and ATM
kinase inhibitor (ATMi; KU-55933) used were from Kudos Pharmaceuticals
(Cambridge, United Kingdom).
Cell lines. The human colon carcinoma HCT116, cervix carcinoma HeLa, and
leukemic Jurkat cell lines were obtained from ATCC (Manassas, VA). HCT116
cells complemented with Mre11 (HCT116-Mre11 cells) were generated in our
laboratory (59). Bax?/?, Bax?/?, Chk2?/?, p53?/?, and p53?/?HCT116 cells
were kind gifts from Bert Vogelstein (Johns Hopkins Oncology Center, Balti-
more, MD) (4, 24, 64). The glioma M059J-Fus1 and M059J-Fus9 cell lines were
from Janice Pluth (Lawrence Berkeley National Laboratory, Berkeley, CA) (21).
PrEC human prostate epithelial cells were obtained from Lonza Walkersville,
Inc. (Walkersville, MD).
Western blotting and antibodies. Cells were washed twice in PBS (phosphate-
buffered saline) and lysed at 4°C in buffer containing 1% SDS (sodium dodecyl
sulfate) and 10 mM Tris-HCl, pH 7.4, and supplemented with protease inhibitors
(Roche Applied Science, Indianapolis, IN) and phosphatase inhibitors (Sigma
Chemical Co., St. Louis, MO). The viscosity of the samples was reduced by brief
sonication. Equal amounts of proteins were boiled for 5 min in Tris-glycine-SDS
sample buffer (Invitrogen, Carlsbad, CA) or heated at 70°C for 10 min in lithium
dodecyl sulfate sample buffer (Invitrogen), separated by Tris-glycine or Tris-
acetate polyacrylamide gel electrophoresis (Invitrogen), and electroblotted onto
nitrocellulose membranes (Bio-Rad, Hercules, CA). The membranes were sat-
urated with milk, incubated overnight at 4°C with primary antibodies, washed,
and then incubated for 45 min with secondary antibodies, i.e., peroxidase-con-
jugated goat anti-mouse or anti-rabbit immunoglobulin G (Santa Cruz Biotech-
nology, Santa Cruz, CA). Signals were revealed by autoradiography with the
enhanced chemiluminescence detection kit (Pierce, Rockford, IL).
The primary antibodies used were anti-P-ATM S1981 (4526; Cell Signaling,
Danvers, MA), anti-53BP1 (NB100-305; NOVUS Biologicals, Littleton, CO),
anti-P-Cdc25A S123 (AP3045a; ABGENT, San Diego, CA), anti-Cdc25A (sc-
7389; Santa Cruz Biotechnology), anti-P-Cdc25C S216 (9528; Cell Signaling),
anti-P-Chk1 S345 (2341; Cell Signaling), anti-Chk1 (sc-8408; Santa Cruz Bio-
technology), anti-P-Chk2 T68 (2661; Cell Signaling), anti-Chk2 (sc-17747; Santa
Cruz Biotechnology), anti-P-DNA-PK S2056 (ab18192; Abcam, Cambridge,
MA), anti-P-DNA-PK T2609 (ab18356; Abcam), anti-DNA-PK (NA57; Calbio-
chem, EMD Biosciences, San Diego, CA), anti-P-E2F1 S364 (ab5391; Abcam),
anti-E2F1 (sc-251; Santa Cruz Biotechnology), anti-?-H2AX (05-636; Upstate,
Temecula, CA), anti-H2AX (07-627; Upstate), anti-histone H3 trimethyl K9
(ab8898; Abcam), anti-lamin B1 (ab16048; Abcam), anti-p21 (OP64; Calbio-
chem), anti-P-p53 S15 (9284; Cell Signaling), anti-P-p53 S20 (9287; Cell Signal-
ing), anti-p53 (sc-126; Santa Cruz Biotechnology), and antitubulin (MS-581; Lab
Vision, Fremont, CA).
Short interfering RNA (siRNA). siRNA targeting DNA-PK was obtained from
Dharmacon, Chicago, IL (SMARTpool, catalogue number M-005030-01).
siRNA targeting Chk2 was obtained from Qiagen, Valencia, CA (validated
siRNA, catalogue number SI02224271). siRNA targeting H2AX and negative
control siRNA were obtained from Ambion, Austin, TX (catalogue numbers
AM16210 and 4635). Cells were seeded in six-well plates, at a density of 200,000
cells per well, 16 h before transfection. For each sample, 500 pmol of siRNA was
mixed with 250 ?l of Opti-MEM (mixture A; Invitrogen). Five microliters of
Lipofectamine 2000 (Invitrogen) was mixed with 250 ?l of Opti-MEM and
incubated for 5 min at room temperature (mixture B). After mixing (mixtures A
and B) and further incubation for 20 min at room temperature, the siRNA/
Lipofectamine complexes were added to 2 ml of culture medium. After 5 h, the
medium was replaced with regular medium and the cells were incubated for a
further 48 h.
Morphological analyses with Hoechst 33342. Cells (105) were washed in PBS,
collected by centrifugation at 500 ? g for 10 min, and resuspended in 500 ?l of
PBS containing Hoechst 33342 (final concentration, ?2 to 10 ?g/ml). After a
30-min incubation in the dark at 37°C, the cells were centrifuged at 500 ? g for
5 min, resuspended in 20 ?l of PBS, and analyzed by fluorescence microscopy.
Immunofluorescence microscopy. Cells were washed with PBS, fixed with 2%
formaldehyde in PBS for 20 min, washed with PBS, postfixed and permeabilized
with cold (?20°C) 70% ethanol for 20 min, washed with PBS, blocked with 8%
bovine serum albumin (BSA) in PBS for 1 h, washed with PBS, incubated with
the first antibody (P-Chk2 T68, 1/500 dilution; ?-H2AX, 1/800 dilution; P-ATM
S1981, 1/250 dilution; P-DNA-PK T2609, 1/250 dilution; H3K9me3, 1/250 dilu-
tion; lamin B1, 1/500 dilution; 53BP1, 1/500 dilution) in 1% BSA in PBS for 2 h,
washed with PBS, incubated with the secondary antibody conjugated with Alexa
Fluor 488 or 568 for 1 h at room temperature, washed with PBS, and mounted
with Vectashield mounting medium with DAPI (4?,6?-diamidino-2-phenylindole)
or propidium iodide to counterstain the DNA (Vector Laboratories). Incubation
with 0.5 mg/ml RNase A for 15 min was performed before the addition of
propidium iodide-containing Vectashield. Confocal images were sequentially
acquired with Zeiss AIM software on a Zeiss LSM 510 NLO confocal system
(Carl Zeiss Inc., Thornwood, NY). Three-dimensional (3D) pictures were real-
ized with the Bitplane (Zurich, Switzerland) Imaris software v6.0. The line
intensity profiles were realized with the Zeiss AIM software v3.2.
?-H2AX quantification by flow cytometry. Three hundred thousands cells were
fixed and permeabilized with cold (?20°C) 70% ethanol overnight, washed with
1% BSA–PBS, and further permeabilized with 0.25% Triton in 1% BSA–PBS for
5 min on ice, incubated with anti-?-H2AX antibody (dilution, 1/37.5) in 10%
normal goat serum–1% BSA–PBS, washed with 1% BSA–PBS, and incubated
with a secondary antibody conjugated with Alexa Fluor 488. Finally, propidium
iodide (final concentration, 0.05 mg/ml) and RNase A (final concentration, 0.5
mg/ml) were added. Fluorescence intensities were determined with a FACScan
flow cytometer (Becton Dickinson) and quantified with CellQuest software (Bec-
Clonogenic assays. Following 1 h of treatment with TRAIL (0.1 ?g/ml), cells
were trypsinized, washed in PBS, and seeded in triplicate at three densities, 100,
1,000, and 10,000 cells per well, in six-well plates. Colonies were allowed to grow
for 10 days and visualized following washing with PBS, fixation in methanol for
30 min, washing again with PBS, and staining with 0.05% methylene blue for 30
min. Percent survival was normalized to the observed number of colonies gen-
erated from untreated cells.
Caspase activity assays. Cells were washed twice in PBS, lysed in 150 mM
NaCl–50 mM Tris-HCl (pH 8.0)–0.1% SDS–1% Nonidet P-40–0.5% sodium
deoxycholate for 30 min at 4°C, and centrifuged (10,000 ? g at 4°C). Fifteen
micrograms of proteins from the resulting supernatant was incubated in 100 mM
HEPES (pH 7.0)–1 mM EDTA–0.1% 3-[(3-cholamidopropyl)-dimethylammo-
nio]-1-propanesulfonate (CHAPS)–10% glycerol–20 mM dithiothreitol in the
presence of the fluorogenic peptide substrate Z-VDVAD-AFC (Z-Val-Asp-Val-
Ala-Asp-AFC, caspase 2), Ac-DEVD-AFC (Ac-Asp-Glu-Val-Asp-AFC, caspase
3), Z-IETD-AFC (Z-Ile-Glu-Thr-Asp-AFC, caspase 8), or Ac-LEHD-AFC (Ac-
Leu-Glu-His-Asp-AFC, caspase 9) (Calbiochem) at 100 ?M. The 7-amino-4-
trifluoromethylcoumarin (AFC) released from the substrate was excited at 400
nm to measure emission at 505 nm. Fluorescence was monitored continuously at
37°C for 30 min in a dual-luminescence fluorimeter (SpectraMax Gemini XS;
Molecular Devices). Caspase activities were determined as initial velocities ex-
pressed as relative intensity per minute per milligram of protein.
DNA fragmentation by filter elution assay. The assay has been described
previously (57). Briefly, cells were incubated with [2-14C]thymidine (0.02 ?Ci/ml)
for 2 days and chased overnight in radioisotope-free medium. After drug treat-
ment, cells were loaded onto a protein-adsorbing filter (Metricel membrane
filter, 0.8-?m pore size, 25-mm diameter; PALL Corp., Cortland, NY), washed
with PBS, and lysed in 0.2% sodium Sarkosyl–2 M NaCl–0.04 M EDTA, pH 10.
The filters were then washed with 0.02 M EDTA, pH 10. DNA was depurinated
by incubation of the filters in 1 M HCl at 65°C and then released from the filters
with 0.4 M NaOH at room temperature. Radioactivity was counted by liquid
scintillation spectrometry in each fraction (wash, lysis, EDTA wash, and filter).
DNA fragmentation was measured as the fraction of disintegrations per minute
in the wash plus lysis fraction plus EDTA wash relative to the total intracellular
disintegrations per minute.
Sub-G1analysis. Cells were washed with PBS, fixed, and permeabilized with
cold (?20°C) 70% ethanol overnight. The next day, cell pellets were washed
VOL. 29, 2009 Chk2 AND ?-H2AX IN TRAIL-INDUCED APOPTOSIS69
again with PBS, resuspended in PBS buffer containing 0.2% NP-40 and 0.5 mg/ml
RNase A, incubated at room temperature for 15 min, and put on ice 10 min prior
the addition of 50 ?g/ml propidium iodide. DNA content was determined with a
FACScan flow cytometer (Becton Dickinson) and quantified with CellQuest
software (Becton Dickinson).
TRAIL induces rapid phosphorylation of the DDR proteins
Chk2, ATM, histone H2AX, and DNA-PK. Figure 1 shows that
FIG. 1. Activation of Chk2, ATM, H2AX, and DNA-PK in response to TRAIL treatment. (A) Kinetics of the effects of TRAIL on DDR proteins
in HCT116 cells. Cells were treated with 0.1 ?g/ml TRAIL for the indicated times, and the indicated proteins were examined by Western blotting. The
phosphospecific antibodies used are listed in Materials and Methods. Tubulin was used as a loading control. The asterisk corresponds to an unspecific
cross-reactive protein for the phospho-T68-Chk2 antibody (P-Chk2 T68). Positive controls (?): P-p53-S20, HCT116 cells 1 h after 20 Gy; p21, HCT116
cells 20 h after 20 Gy; P-E2F1-S364, HT29 cells treated with etoposide (50 ?M, 6 h); P-Chk1-S345, HT29 cells treated with camptothecin (1 ?M, 0.5 h).
(B) Quantification of apoptosis (by Hoechst staining) and DNA fragmentation (measured by filter elution assay). The pound sign for percent apoptosis
at 20 h indicates an underestimation due to loss of signal due to dissolution of nuclei in advanced apoptotic cells. (C) Kinetics of the effects of TRAIL
on Chk2, ATM, H2AX, and DNA-PK phosphorylations in HCT116 cells with Mre11 stable complementation (HCT116-Mre11). Cells were treated with
0.1 ?g/ml TRAIL for the indicated times. Protein phosphorylations (P-Chk2 T68, P-ATM S1981, ?-H2AX, and P-DNA-PK T2609) were analyzed by
Western blotting. Tubulin was used as a loading control. The percentage of apoptosis measured by Hoechst staining is indicated. The pound sign for
percent apoptosis at 20 h indicates an underestimation due to dissolution of nuclei in advanced apoptotic cells. (D) Phosphorylation of histone H2AX
(?-H2AX) in response to TRAIL in HCT116 cells and in HCT116-Mre11 cells.?-H2AX was analyzed by Western blotting. Tubulin was used as a loading
control. (E) Activation of Chk2 in response to TRAIL in HCT116 cells and in HCT116-Mre11 cells. Chk2 phosphorylated on threonine 68 was analyzed
by Western blotting. Tubulin was used as a loading control. (F) Concentration-dependent activation of Chk2, ATM, H2AX, and DNA-PK by TRAIL.
HCT116-Mre11 cells were treated with the indicated TRAIL concentrations for 4 h. Protein phosphorylations (P-Chk2 T68, P-ATM S1981, ?-H2AX,
and P-DNA-PK T2609) were analyzed by Western blotting. Tubulin was used as a loading control. The percentage of apoptosis measured by Hoechst
staining is indicated. The asterisk corresponds to an unspecific cross-reactive protein for the phospho-T68-Chk2 antibody (P-Chk2 T68).
70 SOLIER ET AL.MOL. CELL. BIOL.
(34). Our present study provides no evidence for Chk1 impli-
cation in TRAIL-induced apoptosis (Fig. 1A). Also, inhibition
of ATM and DNA-PK was sufficient to suppress completely
the activation of Chk2 and H2AX (Fig. 6F). Together, these
data suggest that ATM, DNA-PK, and Chk2 are the main
kinases involved in the TRAIL-induced response, contrary to
ATR and Chk1. Nevertheless, we cannot exclude a potential
involvement of ATR. Even if Chk1 was not phosphorylated,
other proteins could be targeted by ATR.
The observed cross talk between ATM and DNA-PK fol-
lowing TRAIL treatment is worth noting because it appears to
be mutual rather than limited to only one direction in which
DNA-PK is phosphorylated by ATM (10). Our present finding
that ATM can be phosphorylated on S1981 in a DNA-PK-
dependent manner (Fig. 7A) is novel. An effect of DNA-PK on
ATM has nevertheless been suggested by some prior studies.
In glioblastoma cells deficient for DNA-PK (M059J), the ATM
protein levels are low (8, 16). In addition, the V3 radiosensitive
CHO cell line presents low ATM protein levels, and when the
amount of DNA-PK is restored by transfection, the levels of
ATM protein are also restored (41). In murine cells with dif-
ferent degrees of DNA-PK deficiency, ATM protein is reduced
From a therapeutic standpoint, our findings suggest that the
levels of Chk2 and activated P-Chk2-T68 in tumor tissues
could have a prognostic value for predicting the efficiency of a
TRAIL therapy. Moreover, our results provide a rationale for
combining TRAIL with DNA-damaging agents. Indeed, DNA
damage (IR or DNA-targeted drugs) might sensitize tumor
cells to TRAIL by preactivating Chk2 and the positive feed-
back loop that amplifies TRAIL-induced apoptosis. Such a
mechanism may partly account for the known synergism be-
tween TRAIL and DNA-damaging therapies (47, 61). Chk2
activation in some human tumors and precancerous lesions (3,
13, 18) could also delay or prevent cancer development, rein-
forcing the importance of an efficient DDR.
This research was supported in part by the Intramural Research
Program of the NIH, National Cancer Institute, Center for Cancer
We thank Susan Garfield (Laboratory of Experimental Carcinogen-
esis, Center for Cancer Research, NCI) for outstanding technical as-
sistance for the generation of 3D microscopy pictures. We also thank
Giovanni Capranico for editorial comments.
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