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: firstname.lastname@example.org.
?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.
treatment with TRAIL induces rapid phosphorylation of Chk2
(at threonine 68), ATM (at serine 1981), H2AX (at serine
139), and DNA-PK (at threonine 2609), starting at 1 h after
TRAIL treatment. These phosphorylations peaked approxi-
mately 2 to 4 h after the addition of TRAIL, concurrently with
apoptosis and the development of DNA fragmentation (Fig.
1B), and then tended to decrease (Fig. 1A).
To determine whether the activations of ATM and Chk2 by
TRAIL were part of a full DDR, we looked at the activation of
other DDR targets of Chk2 and ATM. Cdc25C, which is im-
plicated in entry into G2, was phosphorylated on serine 216,
and Cdc25A, which is required for progression from G1to the
S phase of the cell cycle, was degraded in response to TRAIL
(Fig. 1A). p53 was also phosphorylated on serine 15 (an ATM,
DNA-PK, and ATR substrate) in response to TRAIL but not
on serine 20 (a Chk2 substrate), and its basal levels remained
unchanged (Fig. 1A). p21WAF1/CIP1, one of the p53 transcrip-
tional targets, was not upregulated but was rapidly cleaved
(into an ?14-kDa fragment) in response to TRAIL (Fig. 1A)
(40). In contrast, E2F1 phosphorylation was not detectable and
E2F1 basal levels remained unchanged in TRAIL-treated cells
(Fig. 1A). Moreover, activation of the other checkpoint kinase
Chk1, which also phosphorylates some of the Chk2 substrates,
was not detectable as measured by lack of Chk1 phosphoryla-
tion on serine 345 (Fig. 1A). Together, these results indicate
that TRAIL induces rapid activation of the Chk2/ATM/
DNA-PK pathways without detectable activation of the E2F1,
p21WAF1/CIP1, or Chk1 pathways.
Because HCT116 cells are deficient in Mre11 and in the
MRN complex (a heterotrimer composed of Mre11, Rad50,
and Nbs1) (15, 17) and because MRN binds DSB sites and
activates the ATM and Chk2 checkpoint kinases (30), we ex-
amined whether Mre11 complementation (which also restores
Rad50 and Nbs1 protein levels) (59) could have an impact on
TRAIL-induced DDR activation. Using HCT116 cells stably
complemented for Mre11 (HCT116-Mre11 cells), we previ-
ously showed that Mre11 complementation restores Rad50
and Nbs1 levels and enables robust Chk2 activation by ATM
(59). Figure 1C shows that phosphorylations of Chk2, ATM,
H2AX, and DNA-PK were induced earlier in HCT116-Mre11
cells than in wild-type HCT116 cells in response to TRAIL
(see also Fig. 1D and E). The apoptotic response was also
faster in the Mre11-complemented cells (compare lower panel
C to panel B in Fig. 1), and the phosphorylations of ATM,
Chk2, DNA-PK, and H2AX were observed at TRAIL concen-
trations producing an apoptotic response (Fig. 1A, B, and F).
Thus, activation of ATM, Chk2, DNA-PK, and H2AX in re-
sponse to TRAIL appears to involve MRN.
Since at early times (1 to 2 h), only a fraction of the HCT116
cells underwent apoptosis (Fig. 1B) and H2AX phosphoryla-
tion, we examined whether those cells that were engaging in
early apoptosis were in a specific cell cycle phase. This was
done by flow cytometry with the anti-?-H2AX antibody while
measuring DNA content with propidium iodide. ?-H2AX
formed at all phases of the cell cycle, although with a prefer-
ence for S-phase cells (Fig. 2A). The preferential sensitivity of
S-phase cells to TRAIL is consistent with recent studies (47).
However, the induction of apoptosis by TRAIL in S-phase cells
is probably not directly related to DNA replication since this
induction was not affected by pretreatment with the DNA
polymerase inhibitor aphidicolin (data not shown).
Activation of the DDR pathway was not limited to HCT116
colon carcinoma cells, as it was also induced in all of the other
TRAIL-sensitive human cancer cell lines examined (HeLa cer-
vical carcinoma cells [Fig. 2B], Jurkat T-cell leukemia cells
[Fig. 2C], and M059J glioblastoma cells [see Fig. 6D]), as well
as in normal prostate epithelial cells (Fig. 3C).
Confluent ?-H2AX nuclear staining in response to TRAIL.
Immunofluorescence confocal microscopy revealed chromatin
alterations within 1 h of TRAIL exposure (Fig. 3A). ?-H2AX
staining in those TRAIL-responsive cells was noticeably dif-
ferent from the DDR induced by DNA-damaging agents. In-
stead of the well-defined foci distributed throughout the nu-
cleus and characteristic of ionizing radiation (IR) (Fig. 3B; see
also Fig. 4F) and DNA-damaging drugs (42), ?-H2AX staining
tended to be confluent and followed a progression that could
be subdivided into three phases (Fig. 3A and B). First,
?-H2AX appeared at the nuclear periphery (Fig. 3A, middle
panels; type I, ring staining in panel B). Then the nuclei ap-
peared fully stained without alteration of their overall shape or
size (type II, panstaining in Fig. 3B). Finally, the nuclei shrank
and formed apoptotic nuclear bodies that remained fully and
brightly stained with ?-H2AX (Fig. 3A, lower panels; type III
staining in panel B). Several control experiments were per-
formed to demonstrate that the staining observed with the
?-H2AX antibody was attributable to ?-H2AX. H2AX down-
regulation with siRNA resulted in a 60% reduction in
?-H2AX-positive cells compared to cells transfected with con-
trol siRNA (data not shown). Moreover, using the same anti-
body, we found no ?-H2AX staining in H2AX knockout cells
after IR, contrary to the foci observed in H2AX-proficient cells
(data not shown). In those H2AX knockout fibroblasts, we
could not use TRAIL to induce ?-H2AX because normal mu-
rine embryo fibroblasts were not responsive to TRAIL. Finally,
Western blotting showed only one band with the electro-
phoretic migration expected for ?-H2AX.
To determine the specificity of the ?-H2AX response to
TRAIL for cancer cells, we also examined normal diploid
human cells. Figure 3C demonstrates a similar peripheral and
confluent activation of ?-H2AX in primary human prostate
epithelial cells. However, only a small fraction of those cells
were activated by TRAIL. ?-H2AX staining concerned less
than 10% of the epithelial cells, even upon incubation for
extended times (quantitation in Fig. 3D). The appearance of
?-H2AX after TRAIL treatment was not detectable by West-
ern blotting (Fig. 3D), demonstrating the high sensitivity of the
immunofluorescence method. Together, these results demon-
strate the conservation of the peripheral nuclear ?-H2AX in-
duction (?-H2AX ring staining) induced by TRAIL in normal
To show that the ?-H2AX pattern was inside rather than
around the nucleus, additional double-staining experiments
were performed. Figure 4A and B shows colocalization of
?-H2AX with the heterochromatin fraction at the periphery of
the nucleus (histone H3 trimethyl K9 staining). However,
?-H2AX did not localize with the heterochromatin in the nu-
clear interior. Double staining with lamin B1 (Fig. 4C to E)
also demonstrated that the ?-H2AX pattern was inside the
nucleus (see representative tracing in Fig. 4E). Notably, the
VOL. 29, 2009Chk2 AND ?-H2AX IN TRAIL-INDUCED APOPTOSIS71
nuclear distribution of 53BP1, a protein known to be recruited
with ?-H2AX during IR-induced DSB (Fig. 4F, lower panels),
was not mobilized within the ?-H2AX ring by TRAIL treat-
ment, which is indicative of the different processes leading to
?-H2AX induction by IR and TRAIL (Fig. 4F, middle panels).
Together, these results demonstrate that the induction of
TRAIL-induced apoptosis leads to a rapid DDR activation
starting with the accumulation of phosphorylated histone
H2AX (?-H2AX) in the peripheral heterochromatic region of
the nucleus, which we refer to as ?-H2AX ring staining.
Involvement of Bax and caspases in the activation of the
DDR by TRAIL. Because TRAIL initiates apoptosis at the
plasma membrane rather than by inducing direct DNA dam-
age, we determined whether blocking apoptosis downstream
from the TRAIL receptor could affect the DDR. First, we
examined Bax, a proapoptotic member of the Bcl-2 family
known to be required for TRAIL-induced apoptosis following
its translocation to mitochondria (12, 28, 52) and the release of
Smac/DIABLO, which antagonizes caspase-inhibiting IAP
family proteins (12). In these experiments, we compared
HCT116 wild-type cells (which are Bax?/?) to their Bax knock-
out counterparts (Bax?/?HCT116 cells) (64). Bax?/?cells are
resistant to TRAIL-induced apoptosis (52) and did not show
detectable phosphorylation of the DDR proteins (Fig. 5A and
B), indicating that Bax is required for the induction of those
phosphorylations. Also, pretreatment with the broad-spectrum
caspase inhibitor Z-VAD-fmk abolished TRAIL-induced
phosphorylation of Chk2, ATM, H2AX, and DNA-PK (Fig. 5C
and D). These experiments indicate that DDR activation is an
integral part of the apoptotic process(es) induced by TRAIL.
In response to TRAIL, DNA-PK is the primary kinase for
?-H2AX, whereas both ATM and DNA-PK phosphorylate
Chk2. Since we observed that both ATM and DNA-PK were
activated in response to TRAIL, and both kinases are known
to phosphorylate H2AX and Chk2 (5, 15, 31, 38), we deter-
mined whether ATM and DNA-PK had overlapping or distinct
roles in H2AX and Chk2 phosphorylations. Pretreatment with
Nu7441, a specific DNA-PKi (27, 65), reduced the TRAIL-
induced ?-H2AX response but did not affect apoptosis (Fig.
6A). In contrast, the ATMi KU-55933 (19) had no effect on
either H2AX phosphorylation or cell death (Fig. 6B and F).
Flow cytometry analyses confirmed the reduction of ?-H2AX-
positive cells by the DNA-PKi (Fig. 6C). To further demon-
strate that DNA-PK is critical for H2AX phosphorylation in
FIG. 2. Effect of TRAIL on DDR proteins is observed at all phases of the cell cycle and is not restricted to HCT116 cells. (A) Relationships
between ?-H2AX cellular content and cell cycle. Untreated (upper left panel) and TRAIL-treated (0.1 ?g/ml, 2 h) HCT116 cells (upper right
panel) were analyzed by FACScan flow cytometry. The x axis indicates the DNA content (as determined by propidium iodide staining), and the
y axis indicates ?-H2AX content (cells positive for ?-H2AX are in red, and the negative cells are in gray). Results are quantitated in the lower panel.
(B) Effects of TRAIL on Chk2, ATM, H2AX, and DNA-PK phosphorylations in HeLa cells. Protein phosphorylations were analyzed by Western
blotting. The asterisk corresponds to an unspecific cross-reactive protein for the phospho-T68-Chk2 antibody (P-Chk2 T68). Tubulin was used as
a loading control. The percentage of apoptosis measured by Hoechst staining is indicated at the bottom. (C) Activation of Chk2 in response to
TRAIL in Jurkat cells. Cells were treated with 0.1 ?g/ml TRAIL for the indicated times, and the phosphorylation of Chk2 on T68 was analyzed
by Western blotting. Tubulin was used as a loading control. The percentage of apoptosis measured by Hoechst staining is indicated.
72SOLIER ET AL.MOL. CELL. BIOL.
the TRAIL pathway, we used cells genetically altered for
DNA-PK. In M059J cells complemented with DNA-PK
(M059J-Fus1) (15, 21), TRAIL strongly induced ?-H2AX
while having almost no effect on DNA-PK-deficient M059J
cells (M059J-Fus9) (Fig. 6D). A similar result was observed in
HCT116 cells in which DNA-PK had been knocked down by
siRNA (Fig. 6E). Because it was recently reported that the
JNK inhibitor SP-600125 was able to prevent ?-H2AX forma-
tion in the apoptotic response induced by UVA (32), we tested
SP-600125 in TRAIL-treated cells. We found that SP-600125
(20 or 200 ?M) did not affect ?-H2AX induction by TRAIL
(data not shown). In addition, SP-600125 did not modify
TRAIL-induced apoptosis (data not shown). Together, the
results of the above experiments demonstrate that DNA-PK is
the primary kinase for ?-H2AX in response to TRAIL.
To identify the kinase(s) involved in T68-Chk2 phosphory-
lation, we again tested the DNA-PKi and ATMi. Neither was
able to block T68-Chk2 phosphorylation by itself (Fig. 6A, B,
and F). Pretreatment with SP-600125 (a JNK inhibitor) also
had no effect (data not shown). In contrast, combination of the
ATMi and DNA-PKi suppressed T68-Chk2 phosphorylation
(Fig. 6F). Therefore, we conclude that both ATM and
DNA-PK are implicated in the phosphorylation of Chk2 at T68
in response to TRAIL whereas DNA-PK, but not ATM, is
involved in H2AX phosphorylation at S139.
Cross talk between ATM and DNA-PK in response to
TRAIL. ATM has recently been shown to phosphorylate
DNA-PK at T2609 after IR (10), indicative of cross talk be-
tween ATM and DNA-PK. After TRAIL treatment, we ob-
served that phosphorylation of ATM on S1981 was reduced by
KU-55933 (ATMi), which was expected since S1981 is a known
ATM autophosphorylation site (Fig. 7A) (1). However, S1981-
ATM phosphorylation was also completely suppressed by
Nu7441 (DNA-PKi) (Fig. 7A). Conversely, phosphorylation of
DNA-PK on T2609 was suppressed strongly by ATMi and
completely by DNA-PKi. In contrast, phosphorylation of
DNA-PK on S2056 was unmodified by the ATMi and sup-
pressed only by the DNA-PKi (Fig. 7A). To gain further evi-
dence for the DNA-PK-dependent phosphorylation of ATM,
we used siRNA against DNA-PK. Decreasing the levels of
ylation of ATM on S1981 after TRAIL treatment (Fig. 7B,
FIG. 3. Analyses of ?-H2AX responses to TRAIL. (A) ?-H2AX confocal immunofluorescence staining in HCT116 cells treated with TRAIL.
Images are representative of cells treated with 0.1 ?g/ml TRAIL for 1 or 2 h. ?-H2AX was labeled in green, and nuclei were stained in red with
propidium iodide (PI). (B) Single-cell analyses showing typical ?-H2AX patterns. From left to right: untreated cell, irradiated cell, and cells treated
with TRAIL. The graph shows the relative distribution of the different ?-H2AX patterns. HCT116 cells were treated with 0.1 ?g/ml TRAIL for 1
or 2 h. White columns correspond to peripheral nuclear staining (ring pattern, I), gray columns correspond to panstaining (flooded pattern, II), and black
columns correspond to apoptotic bodies fully stained with ?-H2AX (III). The distribution of the three ?-H2AX patterns was significantly different for
the 1- and 2-h TRAIL treatments (chi-square test, P ? 0.05). (C) ?-H2AX confocal immunofluorescence staining in PrEC cells treated with TRAIL (0.1
control. The percentage of ?-H2AX-positive cells (determined by immunofluorescence microscopy) is indicated for each time point.
VOL. 29, 2009 Chk2 AND ?-H2AX IN TRAIL-INDUCED APOPTOSIS73
FIG. 4. Peripheral distribution of nuclear ?-H2AX in response to TRAIL (0.1 ?g/ml for 1 h). (A) ?-H2AX and heterochromatin confocal
immunofluorescence staining in HCT116 cells treated with TRAIL. Heterochromatin was labeled with histone H3 trimethyl K9 antibody (green),
and ?-H2AX was labeled in red. (B) Relative distribution of ?-H2AX and heterochromatin in a single cell positive for ?-H2AX. Top: confocal
microscopy image. Bottom: intensity tracing. (C) ?-H2AX and lamin B1 confocal immunofluorescence staining in HCT116 cells treated with
TRAIL. (D) Representative 3D image of a ?-H2AX-positive cell double stained with ?-H2AX (red) and lamin B1 (green). Note that the green
signal intensity has been reduced to visualize the ?-H2AX signal. (E) Relative distribution of ?-H2AX and lamin B1 in a single cell positive for
?-H2AX. Top, confocal microscopy image; bottom, intensity tracing. (F) Differential staining patterns for 53BP1 (green) and ?-H2AX (red) in
response to TRAIL and IR (3 Gy) in HCT116 cells. AU, arbitrary units.
74 SOLIER ET AL.MOL. CELL. BIOL.
upper panel). From these results we conclude that, after
TRAIL treatment, ATM phosphorylation on S1981 is under
the control of DNA-PK. We also conclude that DNA-PK phos-
phorylation on T2609 is partially dependent on ATM, while
DNA-PK phosphorylation on S2056 is an autophosphoryla-
tion. Thus, TRAIL induces cross phosphorylations between
ATM and DNA-PK.
We then performed immunofluorescence microscopy exper-
iments to determine whether the three phosphorylated/acti-
vated kinases (ATM, DNA-PK, and Chk2) and ?-H2AX were
colocalized in cells responding to TRAIL. Figure 7C and D
show that cells positive for P-Chk2-T68 were also positive for
?-H2AX, P-ATM-S1981, and P-DNA-PK-T2609. Moreover,
these proteins colocalized at the periphery of the nucleus (ring
staining) as described above for ?-H2AX (Fig. 3A and B).
Control experiments demonstrated that the staining observed
with the P-Chk2-T68 antibody was attributable to P-Chk2-T68.
Indeed, Chk2 downregulation by siRNA resulted in 80% fewer
P-Chk2-T68-positive cells compared to cells transfected with a
control siRNA (data not shown). Moreover, using the same
antibody, we found no P-Chk2-T68 staining in Chk2-deficient
HCT15 cells after IR, contrary to the foci observed in Chk2-
proficient cells (data not shown). The absence of P-Chk2-T68
staining (with the same antibody) after camptothecin treat-
ment in Chk2-deficient HCT15 cells has also been published
recently (44). Together, our findings demonstrate that TRAIL
induces the coincident activation of ATM, DNA-PK, Chk2,
and histone H2AX as individual cells induce DDR and that
these phosphorylated proteins are colocalized in similar chro-
matin regions that define the ring staining that occurs in the
early phase of apoptosis.
Functional impact of Chk2 on TRAIL-induced apoptosis.
Because of our finding that Chk2 was robustly activated by
TRAIL, and of the previously known effect of Chk2 in pro-
moting apoptosis (20, 58), we investigated Chk2’s functional
involvement in TRAIL-induced apoptosis.
Using Chk2 siRNA (Fig. 8A), we first observed that Chk2
downregulation had a negative impact on TRAIL-induced cell
detachment, one of the early events in cells undergoing pro-
grammed cell death. As shown in Fig. 8B (left panels with
representative images and quantitations under images), the
fraction of cells that remained adherent after TRAIL treat-
ment was significantly greater in cells whose Chk2 had been
downregulated by siRNA. Two hours after TRAIL addition,
49% of the Chk2 siRNA cells remained attached while only
16% of the control siRNA cells were attached. That difference
persisted for up to 4 h during TRAIL treatment (Fig. 8B, right
FIG. 5. Bax and caspases are required for the activation of Chk2, ATM, H2AX, and DNA-PK in response to TRAIL. (A) Bax requirement for
TRAIL-induced DDR response. Bax?/?or Bax?/?HCT116 cells were treated with TRAIL as indicated. Protein phosphorylations (P-Chk2 T68,
P-ATM S1981, and ?-H2AX) were analyzed by Western blotting. Tubulin was used as a loading control. (B) Representative immunofluorescence
experiment showing ?-H2AX staining after TRAIL treatment (0.1 ?g/ml, 1 h) in Bax?/?or Bax?/?HCT116 cells. ?-H2AX was labeled in green,
and nuclei were stained in red with propidium iodide. (C) Caspase requirement for TRAIL-induced DDR response. HCT116 cells were treated
with Z-VAD-fmk for 1 h prior to the addition of TRAIL. 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 asterisk corresponds to an unspecific cross-reactive protein for the
P-Chk2-T68 antibody. (D) Representative immunofluorescence experiment showing ?-H2AX and P-Chk2 T68 staining after TRAIL treatment in
HCT116-Mre11 cells pretreated with Z-VAD-fmk. The cells were treated with Z-VAD-fmk at 100 ?M for 1 h prior to exposure to TRAIL at 0.1
?g/ml for 2 h. ?-H2AX and P-Chk2 were labeled in green, and nuclei were stained in red with propidium iodide.
VOL. 29, 2009 Chk2 AND ?-H2AX IN TRAIL-INDUCED APOPTOSIS 75
Next we determined the impact of Chk2 downregulation by
siRNA on cell survival measured by clonogenic assays. Figure
8C shows that Chk2 knockdown significantly increased cell
We also studied the impact of Chk2 on TRAIL-induced
?-H2AX. Figure 8D shows that downregulation of Chk2 de-
layed the progression of ?-H2AX staining (from type I to type
III). These results suggest that Chk2 activation tends to facil-
itate the ?-H2AX response to TRAIL.
Finally, to determine the molecular impact(s) of Chk2
knockdown on TRAIL-induced apoptosis, we measured the
activation of several caspases. Activation of caspases 2, 3, 8,
and 9 was delayed in the Chk2 knockdown cells (Fig. 8E).
Together, the results obtained with siRNA against Chk2
FIG. 6. H2AX is phosphorylated by DNA-PK and Chk2 is phosphorylated by both DNA-PK and ATM in response to TRAIL. (A) Effect of
the DNA-PKi NU7441 on the phosphorylations of Chk2 and H2AX after TRAIL treatment. HCT116 cells were treated with the DNA-PKi (1 h)
prior to the addition of TRAIL. Protein phosphorylations (P-Chk2 T68 and ?-H2AX) were analyzed by Western blotting. Tubulin was used as a
loading control. The percentage of apoptosis measured by Hoechst staining is indicated. (B) Effect of the ATMi KU-55933 on the phosphorylations
of Chk2 and H2AX after TRAIL treatment. HCT116 cells were treated with the ATMi (1 h) prior to the addition of TRAIL. Protein
phosphorylations (P-Chk2 T68 and ?-H2AX) were analyzed by Western blotting. Tubulin was used as a loading control. The percentage of
apoptosis measured by Hoechst staining is indicated. (C) Inhibition of ?-H2AX formation by the DNA-PKi (10 ?M, 1 h) in TRAIL-treated
HCT116 cells (0.1 ?g/ml, 3 h). Cells were analyzed by FACScan flow cytometry. The x axis indicates ?-H2AX content (?-H2AX-positive cells are
circled in gray), and the y axis indicates the number of cells. The percentage of ?-H2AX-positive cells was significantly reduced by pretreatment
with the DNA-PKi (n ? 3; unpaired t test, P ? 0.001). (D) Defective induction of ?-H2AX after TRAIL treatment in Fus9 (without DNA-PK)
compared to Fus1 (with DNA-PK) M059J cells. Cells were treated as indicated. Tubulin was used as a loading control. The percentage of apoptosis
measured by Hoechst staining is indicated. (E) Decreased H2AX phosphorylation in response to TRAIL in cells transfected with siRNA against
DNA-PK. (Upper panel) HCT116 cells were treated with TRAIL (0.1 ?g/ml, 3 h) 48 h after transfection with siRNA against DNA-PK or a negative
control siRNA. ?-H2AX was analyzed by Western blotting. Tubulin was used as a loading control. (Lower panel) Western blotting showing the
efficiency of DNA-PK downregulation by siRNA. Total DNA-PK was analyzed by Western blotting. Tubulin was used as a loading control.
(F) Effects of both ATMi and DNA-PKi on the phosphorylations of Chk2 and H2AX after TRAIL treatment. HCT116 cells were treated with the
ATMi or/and the DNA-PKi (10 ?M, 1 h) prior to the addition of TRAIL. P-Chk2 T68, total Chk2, ?-H2AX, and total H2AX were analyzed by
Western blotting. Tubulin was used as a loading control. The numerical values, obtained by densitometry analysis (ImageQuant software),
represent the ratio of P-Chk2 T68 or ?-H2AX to tubulin.
76 SOLIER ET AL.MOL. CELL. BIOL.
demonstrate that Chk2 affects TRAIL-induced programmed
cell death at several levels: kinetics of caspase activation (Fig.
8E), ?-H2AX response (Fig. 8D), cell detachment (Fig. 8B),
and ultimately cell survival (Fig. 8C).
To further examine the functional role of Chk2 in apoptosis,
we also performed experiments with Chk2 knockout cells (24).
Figure 9A shows that apoptosis measured by sub-G1response
was significantly reduced in Chk2?/?cells.
Finally, based on our finding that TRAIL-induced activation
of the (Chk2/ATM/DNA-PK) DDR pathway invoked a partial
p53 response, as determined by phosphorylation of p53 at S15
(Fig. 1A), the role of p53 in TRAIL-induced cell killing was
FIG. 7. Cross talk between ATM and DNA-PK in response to TRAIL. (A) Effect of the DNA-PKi on ATM phosphorylation on S1981 and
effect of the ATMi on DNA-PK phosphorylation on T2609. HCT116 cells were treated with the ATMi and/or the DNA-PKi for 1 h prior to the
addition of TRAIL. Protein phosphorylations (P-ATM S1981, P-DNA-PK T2609, and P-DNA-PK S2056) were analyzed by Western blotting.
Tubulin was used as a loading control. (B) Decreased ATM phosphorylation on S1981 in response to TRAIL in cells transfected with siRNA
against DNA-PK. (Upper panel) HCT116 cells were treated with TRAIL (0.1 ?g/ml, 3 h) 48 h after transfection with siRNA against DNA-PK or
a negative control siRNA. P-ATM S1981 was analyzed by Western blotting. Tubulin was used as a loading control. (Lower panel) Western blotting
showing the efficiency of DNA-PK downregulation by siRNA. Total DNA-PK was analyzed by Western blotting. Tubulin was used as a loading
control. (C) Immunofluorescence experiment showing the colocalization of P-Chk2 T68 with ?-H2AX, P-ATM S1981, and P-DNA-PK T2609 in
response to TRAIL. HeLa cells were treated with TRAIL at 0.1 ?g/ml for 2 h. ?-H2AX, P-ATM S1981, or P-DNA-PK T2609 was labeled in red,
and P-Chk2 T68 was labeled in green. The dashed circles define the nuclei. (D) Tables showing the relationships between the numbers of cells
positive for P-Chk2 T68, ?-H2AX, P-ATM S1981, and P-DNA-PK T2609. Double-positive, single-positive, and double-negative cells were scored
as shown in panel C. Expected numbers from contingency tables are in parentheses (purple). Results of chi-square tests are shown at the right (in
all three cases, P ? 0.001).
VOL. 29, 2009 Chk2 AND ?-H2AX IN TRAIL-INDUCED APOPTOSIS77
FIG. 8. Functional impact of Chk2 on TRAIL-induced apoptosis. (A) Western blotting of a representative experiment showing the efficiency
of Chk2 downregulation by siRNA. HCT116 cells were transfected with siRNA against Chk2 or negative control siRNA. Total Chk2 was analyzed
by Western blotting. Tubulin was used as a loading control. (B) Effect of Chk2 on cell detachment. HCT116 cells transfected with siRNA against
Chk2 or a negative control siRNA were treated with TRAIL (0.1 ?g/ml) for the indicated times. (Left panels) Representative microscopic pictures
under visible light. Values under the panels correspond to the fractions of attached cells (? standard deviation). (Right panel) The y axis represents
the percentage of attached cells. The cells transfected with negative control siRNA are in black, and those transfected with siRNA against Chk2
are in gray.***, P ? 0.001, unpaired t test; n ? 3. (C) Effect of Chk2 on cell survival. HCT116 cells transfected with siRNA against Chk2 or
negative control siRNA were treated with TRAIL for 1 h (0.1 ?g/ml). Ten thousands cells were then seeded, and clonogenic assays were
performed. Two independent experiments are plotted; the black bars correspond to negative control siRNA, and the gray bars correspond to
78 SOLIER ET AL.MOL. CELL. BIOL.
examined with isogenic p53?/?and p53?/?HCT116 cells (4).
Caspase activation was comparable in p53?/?and p53?/?cells
(data not shown), and attenuation of this caspase response by
Chk2 downregulation was similar in both cell types (Fig. 9B),
indicating that the proapoptotic effect of Chk2 is p53 indepen-
Our study focused on the involvement of Chk2 and histone
H2AX in response to DR activation by TRAIL. We show that
Chk2, following its activation by DNA-PK and ATM, can am-
plify the apoptotic response elicited by TRAIL by a feedback
loop independently of p53. The Chk2-mediated positive feed-
back extends outside the nucleus, as it involves upstream
caspases 8 and 9. Caspase 8 forms a complex with the TRAIL
membrane receptors (DR4 and DR5) within the DISC, and
caspase 9 is a key effector of the apoptosome. Another
TRAIL-induced feedback loop implicating caspase 8 has re-
cently been described, which involves the nuclear translocation
of a self-cleaved death effector domain segment of caspase 8
and p53-dependent upregulation of procaspase 8 gene expres-
sion (62). Our feedback mechanism would act more directly on
caspases than the p53-dependent transcriptional activation of
procaspase 8 (62). Figure 10 gives an outline of the TRAIL-
induced DDR pathways and schematizes the feedback loops
that are revealed in our study.
The functional impact of Chk2 on TRAIL-induced apopto-
sis was detectable as an enhancement of cell detachment, an
increased cell death measured by clonogenic assays, an in-
creased ?-H2AX induction, and a faster activation of caspases
(Fig. 8). In fact, TRAIL was remarkably potent in HCT116
cells. Even after only 1 h of TRAIL exposure, only ?1% of the
cells were able to form colonies (Fig. 8C). Therefore, the cells
progress to apoptosis once they have been committed upon
TRAIL exposure. Inactivation of Chk2 increased the number
of surviving colonies by 40 to 60% (Fig. 8C), demonstrating the
importance of Chk2 in eliminating residual cells. The func-
tional impact of Chk2 may be quite significant because of the
potentially severe physiological consequences of abortive
apoptosis. Apoptosis has been viewed as an important mech-
anism to eliminate oncogenes from dying tumor cells or virus-
infected cells. Also, even when cells die, it is important that
apoptosis proceed to completion to avoid autoimmune dis-
eases, which have been linked to circulating DNA fragments
(39, 63). Thus, it is possible that Chk2 promotes the comple-
tion of the apoptosis program, thereby avoiding the survival of
siRNA against Chk2. The y axis indicates the number of colonies.***, P ? 0.001, chi-square test. (D, upper panels) Representative 3D single-cell
analyses showing the different ?-H2AX patterns (Fig. 3B). (Lower panel) HCT116 cells transfected with siRNA against Chk2 or a negative control
siRNA were treated with 0.1 ?g/ml TRAIL for 1 or 2 h. White columns correspond to peripheral nuclear staining (ring pattern, I), gray columns
correspond to panstaining (flooded pattern, II), and black columns correspond to apoptotic bodies fully stained with ?-H2AX (III). The
distribution of the three ?-H2AX patterns was significantly different for the Chk2 siRNA- and control siRNA-treated cells (chi-square tests, P ?
0.05). (E) Effect of Chk2 on caspase activities. HCT116 cells transfected with siRNA against Chk2 or a negative control siRNA were treated with
TRAIL at 0.1 ?g/ml for 1 to 6 h, and caspase 2, 3, 8, and 9 activities were measured (AU, arbitrary units). The cells transfected with negative control
siRNA are in black, and those transfected with siRNA against Chk2 are in gray.
FIG. 9. Further characterization of the functional impact of Chk2 with HCT116 derivatives. (A) Effect of Chk2 on sub-G1. HCT116 cells or
Chk2?/?HCT116 cells were treated with TRAIL at 0.1 ?g/ml for 3, 6, or 20 h. The y axis represents the percentage of cells with sub-G1DNA.
***, P ? 0.001, unpaired t test; n ? 3. (B) The effect of Chk2 on caspase 3 and 8 activities is independent of p53. p53?/?and p53?/?HCT116
cells transfected with siRNA against Chk2 or a negative control siRNA were treated with TRAIL at 0.1 ?g/ml for 2 h before measurement of
caspase 3 or 8 activity. The y axis represents the reduction of caspase activity by Chk2 siRNA normalized to the control siRNA (percent). p53?/?
HCT116 cells are in gray, and p53?/?HCT116 cells are in white.
VOL. 29, 2009Chk2 AND ?-H2AX IN TRAIL-INDUCED APOPTOSIS79
abnormal cells and the release of toxic metabolic intermedi-
To our knowledge, our study is the first to implicate Chk2 in
TRAIL-induced apoptosis independently of p53. Prior studies
had provided functional evidence for the proapoptotic role of
Chk2 in the context of DNA damage produced by IR, radio-
mimetic agents, and arsenic trioxide (20, 23, 25, 58). In those
models, the proapoptotic function of Chk2 was linked to p53
activation. In contrast, in our study, the proapoptotic function
of Chk2 is p53 independent. p53 was phosphorylated on serine
15 but neither phosphorylated on serine 20 nor stabilized as
determined by lack of p53 protein accumulation in TRAIL-
treated cells (Fig. 1A). Also, p21WAF1/CIP1was not upregulated
by TRAIL (Fig. 1A). These results are in accordance with the
literature showing that phosphorylation on serine 20 is re-
quired for stabilization of p53 (9) and enhancement of the
transcriptional activation of p21WAF1/CIP1
p21WAF1/CIP1was rapidly cleaved in response to TRAIL (Fig.
1A), probably due to caspase 3 activity (40). The degradation
of p21WAF1/CIP1could facilitate apoptosis (36). We also found
that the proapoptotic role of Chk2 was unaffected in the ab-
sence of p53 (Fig. 9). Thus, our findings are consistent with
other studies showing that p53 is not essential for TRAIL-
induced apoptosis (45, 46).
A main focus of our study is the H2AX response to TRAIL.
We show that histone H2AX is rapidly phosphorylated on
serine 139 (?-H2AX) and that ?-H2AX tends to form a con-
fluent staining that initiates in peripheral nuclear regions (?-
H2AX ring staining) before gross changes in nuclear morphol-
ogy that characterize apoptosis (Fig. 3, 4, 5, 7, and 8). We had
previously shown ?-H2AX formation in response to Fas anti-
body, staurosporine, and DNA-damaging agents (50) soon af-
ter the discovery of ?-H2AX (51). But at that time, we did not
investigate the nuclear distribution of ?-H2AX by immunoflu-
orescence microscopy. In the present study, several novel
points are noteworthy regarding TRAIL-induced ?-H2AX ac-
tivation. It is remarkably rapid (starting within 1 h), while the
cells retain an overall normal morphology. Only a fraction of
the cells initially show ?-H2AX staining, and those cells tend to
by p53 (22).
be in S phase, although not exclusively (Fig. 2A). The initial
?-H2AX staining initiates as a confluent pattern at the periph-
ery of the nucleus (?-H2AX ring staining) before diffusing to
form a panstaining pattern covering the entire nucleus and,
later on, the nuclear bodies (Fig. 3, 4, and 8). This confluent
pattern was also observed in normal epithelial cells treated
with TRAIL, although in much fewer cells than in the cancer
cells examined here (Fig. 3C and D), which suggests conser-
vation of the ?-H2AX induction process in both cancer and
normal cells. The ?-H2AX peripheral distribution (?-H2AX
ring staining) occurred within the peripheral heterochromatic
regions at the contact of the lamin B1 nuclear boundary (Fig.
4). The TRAIL-induced ?-H2AX patterns never formed de-
tectable nuclear foci and were not accompanied by the recruit-
ment/colocalization of 53BP1, another ubiquitous DDR factor,
which sets apart the TRAIL-induced ?-H2AX response from
the focal response(s) characteristic of DNA-damaging agents
(49). The ring staining for ?-H2AX also contained activated
ATM phosphorylated on S1981, activated DNA-PK phosphor-
ylated on T2609, and activated Chk2 phosphorylated on T68
(Fig. 7C). Further studies are under way to determine whether
this novel ?-H2AX response is induced during other apoptotic
processes besides TRAIL-induced DR pathway activation.
We found that Chk2 has an impact on the progressive in-
duction of ?-H2AX staining in response to TRAIL. Chk2
siRNA slowed down the formation of ?-H2AX-positive cells
with nuclear segmentation (type III staining) (Fig. 8D). This
reduction could result from delayed caspase activation (Fig.
8E). However, it is also plausible that Chk2 acts directly on
chromatin. In fact, histone H1 is a substrate for Chk2 (67).
Thus, H1 phosphorylation by Chk2 might be part of the chro-
matin modifications controlled by Chk2. This possibility is at-
tractive in the context of recent studies showing that Chk2
activation by DNA damage induces the release of H1.2 from
chromatin and its translocation to the mitochondria, where
H1.2 can act as a positive regulator of apoptosome formation
Our experiments demonstrate that the kinase responsible
for the induction of ?-H2AX by TRAIL is DNA-PK (Fig. 6). In
contrast, Chk2 is phosphorylated by both ATM and DNA-PK
in response to TRAIL. Thus, the ATM and DNA-PK pathways
appear to branch out upstream from Chk2 and H2AX (Fig.
10). Our finding that DNA-PK is the primary ?-H2AX kinase
during TRAIL-induced apoptosis is consistent with another
study showing that DNA-PK is responsible for ?-H2AX for-
mation during staurosporine-induced apoptosis (38). However,
contrary to a study of UVA-induced apoptosis (32), we found
that JNK kinase is not involved in ?-H2AX induction by
TRAIL and that knocking out H2AX by siRNA had no effect
on TRAIL-induced apoptosis (data not shown). These differ-
ences suggest the existence of various nuclear pathways acti-
vated during apoptosis. The general dispensability of H2AX
for the execution of apoptosis is actually consistent with the
viability and normal embryonic development of H2AX knock-
out mice (7).
To investigate the potential involvement of ATR in the
TRAIL pathway, we looked at the activation of Chk1, which is
a preferential ATR substrate. Activation of Chk1 following its
cleavage by caspases has been reported to contribute to Chk1-
dependent apoptosis in response to DNA-damaging agents
FIG. 10. Schematic representation of the caspase- and Bax-depen-
dent activation of DNA-PK, ATM, H2AX, and Chk2 in response to
TRAIL. Open arrows correspond to activation.
80 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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