Carcinogenesis vol.28 no.10 pp.2207–2217, 2007
doi:10.1093/carcin/bgm101
Advance Access publication May 17, 2007
Aspirin blocks proliferation in colon cells by inducing a G1 arrest and apoptosis through
activation of the checkpoint kinase ATM
M.Gloria Luciani, Christoph Campregher and Christoph
Gasche
Division of Gastroenterology and Hepatology, Medical University of Vienna,
KIM4, Währinger Gürtel 18, A-1090 Vienna, Austria
To whom correspondence should be addressed. Tel: þ43 1 404004764;
Fax þ43 1 404004735;
Email: [email protected]
Colorectal cancer (CRC) is the most common gastrointestinal
malignancy. Most of the clinical data on CRC prevention have
come from the use of aspirin. Besides inhibition of cyclooxygenases, aspirin has a diversity of molecular effects that counteract
colon carcinogenesis. Aspirin restrains cell proliferation by inducing a G1 arrest in colorectal cells. To determine which cell cycle
checkpoint pathways are involved in this response, colorectal cell
lines wild-type or defective for p53 and p21Waf1/Cip1 were treated
with aspirin or the anti-proliferative drug sulindac sulfide, then
assayed for proliferative activity, for cell cycle progression and
apoptosis, for the activation and phosphorylation of checkpoint components and for the transcriptional up-regulation of
p21Waf1/Cip1 and Bax. Aspirin and sulindac sulfide induced a G1
arrest within 48 h. While all cell lines responded in a comparable
way to sulindac sulfide, the aspirin-induced G1 arrest was dependent on p21Waf1/Cip1—as cells lacking the cyclin-dependent kinase
inhibitor failed to show this arrest—and on ataxia-telangiectasiamutated kinase (ATM)—as the inhibitor caffeine abrogated the
checkpoint. Moreover, aspirin induced cell death mainly in cells
expressing p53. Aspirin induced the phosphorylation of p53 at
residue Ser15 within 8 h in a caffeine-dependent manner, and also
caused the activation of checkpoint kinase 2 and the cleavage of
caspase 7. Our results suggest that aspirin induces a G1 arrest
and apoptosis by activating p53 and p21Waf1/Cip1 in an ATMdependent way. By activating these checkpoint pathways, aspirin
may restrain uncontrolled proliferation of colorectal cells, enhance their response to stresses such as DNA damage and promote
entry of abnormal cells into apoptosis.
Introduction
Cancer chemoprevention is understood as the use of natural, synthetic
or chemical agents to reverse, suppress or prevent carcinogenic progression to invasive cancer. Findings from epidemiological and clinical studies as well as animal models of colon carcinogenesis imply
that some non-steroidal anti-inflammatory drugs (NSAIDs), salicylate
derivatives and inhibitors of the cyclooxygenase (COX)-2 may be
effective against the development of colorectal cancer (CRC) (1).
Pharmacologically, NSAIDs inhibit COXs in various cell types and
tissues. COX-2 inhibition is believed to underlie the chemopreventive
effect of NSAIDs possibly through reducing cell proliferation, inducing apoptosis or modulating angiogenesis. NSAIDs inhibit the proliferation rate, alter the cell cycle distribution and induce apoptosis in
colon cancer cell lines. Among the most studied agents of the COX-2
inhibitor group of compounds are aspirin and sulindac and derivatives
(2,3). Salicylates were shown to have a chemopreventive role in CRC
Abbreviations: ATM, ataxia-telangiectasia-mutated kinase; ATR, ATM- and
Rad3-related kinase; CDK, cyclin-dependent kinase; Chk2, checkpoint kinase
2; COX, cyclooxygenase; CRC, colorectal cancer; DSB, double-strand break;
MTT, methylthiazolyldiphenyl-tetrazolium bromide; NF-jB, nuclear factor
kappa B; NO, nitric oxide; NSAID, non-steroidal anti-inflammatory drug;
pAb, polyclonal antibody.
through COX-independent mechanisms, such as inhibition of the
transcription factor nuclear factor kappa B (NF-jB) (4). Similar lines
of research suggested that aspirin alters the expression of genes involved in mismatch repair and cell cycle progression (5). All these
pieces of experimental evidence point to the interference of aspirin
with cell cycle progression, although the biochemical pathways involved are still obscure.
To maintain genome stability and monitor the structure of chromosomes, eukaryotic cells have evolved surveillance mechanisms called
cell cycle checkpoints that block cell cycle progression at specific
stages to allow the cell to recover from the impairment (6). Checkpoint pathways comprise damage sensors, signal transducers and effectors. Ataxia-telangiectasia-mutated kinase (ATM) and ATM- and
Rad3-related kinase (ATR) are phosphatidylinositol-3-like kinases
central to the DNA damage response, which can cause activation of
checkpoints, DNA repair and apoptosis (7). ATM is the protein product
of the gene mutated in the multisystem disorder ataxia-telangiectasia,
which is characterized by neuronal degeneration, immunodeficiency,
chromosomal instability and cancer predisposition (8). The ATM
pathway responds to the presence of double-strand breaks (DSBs)
and can be activated during all phases of the cell cycle. In the absence
of a checkpoint mechanism, premature progression through the cell
cycle can be either lethal to the cell or result in oncogenic transformation. A hallmark of the transformed state of tumor cells is incompetent checkpoint control, resulting in accumulation of mutations and
genetic abnormalities (9). When checkpoint control is compromised,
initiation of S phase, or the onset of mitosis, occurs despite cellular
damage, and the ensuing genetic instability may lead to the emergence of a malignant clone. Cells in which checkpoint control is
disrupted are susceptible to the accumulation of additional genotoxic
damage (10).
The tumor suppressor protein p53 is the major known mediator of
the checkpoint-induced arrest in the G1 phase of the cell cycle. A
variety of cellular stresses including DNA damage, hypoxia, nucleotide depletion, viral infection and cytokine-activated signaling pathways transiently stabilize the p53 protein, cause it to accumulate in
the nucleus and activate it as a transcription factor (11). P53 induces
cell cycle arrest, through the cyclin-dependent kinase (CDK) inhibitor
p21Waf1/Cip1 (12), preventing the replication of damaged DNA. Alternatively, p53 can promote apoptosis, mainly through Bax (13), which
is important for eliminating defective cells. Mutations in the p53 gene
occur in half of all human cancers, and regulation of the protein is
defective in a variety of others (14). Stabilization of p53 in response to
ionizing radiation and ultraviolet light is dependent on the ATM and
ATR kinases, respectively (15). ATM is required for homologous recombination and cell cycle checkpoint activation after DNA damage
(16). In addition, germline mutations of ATM lead to radiation hypersensitivity, growth retardation, immunodeficiency and greatly increased cancer risk (17). Upon activation, ATM undergoes Ser1981
auto-phosphorylation and dimer separation (18). As a protein kinase,
ATM functions by phosphorylating and activating a number of DNA
repair and checkpoint proteins including p53 and checkpoint kinase 2
(Chk2), and it has been established previously that ATM is required to
activate a p53-dependent cell cycle G1 arrest upon DNA damage (19).
ATM and ATR both directly phosphorylate p53 in vivo on Ser15 and
Ser37 causing stabilization (20). The ATM and ATR substrate Chk2
phosphorylates p53 on Ser20, which regulates the binding to and
degradation by Mdm2 and thereby stabilizes p53 (21).
Our present study is focused on understanding the mechanisms by
which aspirin exerts its control over cell cycle progression and activates cellular checkpoints in colon cells. We show that aspirin induces
a caffeine-sensitive G1 arrest, which is dependent on p21Waf1/Cip1 and
ATM, and cell death in cells expressing p53. Moreover, aspirin leads
Ó The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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M.G.Luciani et al.
to phosphorylation of p53, c-H2AX and Chk2 in an ATM-dependent
manner, indicating that it activates a cellular checkpoint pathway including these proteins.
Materials and methods
Cell lines
HT29 and HCT116 CRC cells were obtained from American Type Culture Collection. HCT116p53/ cells (p53-null) and HCT116p21/ (p21Waf1/Cip1-null)
were generated in Dr Vogelstein’s laboratory as described (22). Cells were
grown as monolayers in Iscove’s modified Dulbeco’s medium (Gibco/
Invitrogen, Vienna, Austria) containing 2 nM glutamine and 10% fetal bovine
serum at 5% CO2.
Antibodies, plasmids and reagents
Aspirin (Sigma, Vienna, Austria) was dissolved in culture medium at 10 mM
and pH adjusted to 7.2 with NaOH. Sulindac sulfide (Sigma) was dissolved in
DMSO at 400 mM and sterile filtered. Caffeine (Sigma) was dissolved in H2O
and used at 5 mM. Cells were treated at subconfluent densities. All experiments
were performed in triplicate. Total cell lysates were obtained as described (23)
and western blots were performed according to standard procedures (23). Antibodies used were as follows: monoclonal antibody anti-p53 DO7 (Calbiochem,
Vienna, Austria); polyclonal antibody (pAb) anti-phopsho-p53 Ser15 (Cell
Signaling, Frankfurt am Main, Germany); monoclonal antibody anti-p21Waf1/
Cip1
(Cell Signaling); pAb for cleaved caspase 7 (Cell Signaling); monoclonal
antibody anti-ATM (Abcam, Cambridge, UK) and pAb anti-phospho ATM
Ser1981 (Upstate, Vienna, Austria); pAb anti-Chk2 and pAb anti-phospho
Chk2 Thr68 (Cell Signaling) and pAb anti-H2AX and pAb anti-phospho cH2AX Ser139 (Abcam). When needed, HCT116p53/ cells were transfected
with 0.5–1 lg pcDNA-p53 coding for full-length p53 (24) or empty vector
(Invitrogen). pGL3-p21-luciferase and pGL3-Bax-Luciferase constructs were
a gift from M.Oren (Weizmann Institute of Science, Israel) and they are described elsewhere in the text. All transfections were performed with the Effectene reagent (Qiagen, Vienna, Austria) according to the manufacturer’s
instructions.
Cell proliferation assay
Cells were plated in 96-well microtiter plates at a density of 5000 cells per well
in medium. Twenty-four hours later, cells were treated with aspirin (0–10 mM)
or sulindac sulfide (0–400 lM); untreated cells served as controls. After treatment for 48 or 72 h, cell proliferation was assayed using methylthiazolyldiphenyltetrazolium bromide salt (MTT) as described previously (23).
Results
Aspirin reduces the proliferation of human colon cancer cell lines
In order to investigate the proliferation inhibitory effect of aspirin on
colon cells, HCT116 and HT29 were treated with aspirin (0–10 mM)
for 72 h. These cell lines have been chosen on the basis of their
different expression of COX-2, p53 and downstream genes (Figure
S1, supplementary data are available at Carcinogenesis online). In
fact, HCT116 do not express COX-2 (27), but bear a wild-type form
of p53, whereas HT29 express COX-2 but have a mutated p53
(p53R273H). Cellular proliferation was assessed by MTT assay. Aspirin
treatment induced a concentration-dependent reduction in the proliferation rate of both cell lines (Figure 1A). However, 72 h after treatment, the number of viable cells was higher in the HT29 cell line than
in HCT116 (e.g. at 0.62 mM aspirin: 68.2 ± 4.2 versus 45.6 ± 1.6% of
control, P , 0.0001 or at 2.5 mM: 35.6 ± 1.7 versus 23.6 ± 1.0; P ,
0.0001). As HT29 cells express a mutant form of p53 (p53R273H), we
wished to investigate whether the difference in response of the 2 cell
lines was due to an active p53 pathway. The p53-null HCT116p53/
is a cell line isogenetic to the wild-type clone HCT116 created by
targeted homologous recombination [Figure S1 and (28)]. HCT116
cells and HCT116p53/ were incubated in the presence of 0, 2.5, 5
and 10 mM aspirin for 72 h (Figure 1B). We found that HCT116p53/
cells were more resistant to aspirin treatment than HCT116 (p53-wt)
(e.g. at 2.5 mM aspirin 47.6 ± 2.1 versus 31.9 ± 2.4% of control; P ,
0.0001); moreover, re-introduction of p53 into HCT116p53/ cells
via transient transfection (Figure 1D) increased their sensitivity to
aspirin treatment to an extent similar to that of HCT116 cells (Figure
1C). All these results point to a role of p53 for aspirin-mediated
changes in proliferation and/or cell death. The results shown in Figure
1A and B have been normalized to the respective untreated control to
account for the differences in the growth rate of the various colon cell
lines. The corresponding non-normalized graphs, as well as the one in
Figure 2B described here below, are shown as supplementary data are
available at Carcinogenesis online (Figure S2A–D).
Luciferase assay
Cells were seeded in a 6-well culture plate at a density of 105 cells per well and
transfected with 0.5 lg of either pGL3-Basic-Vector (Promega, Mannheim,
Germany; containing no promoter upstream of the Luciferase gene), pGL3Control-Vector (Promega; containing the SV40 constitutive promoter upstream
of the Luciferase gene) or pGL3-based plasmids upstream of the Luciferase
coding sequence ([containing the promoters of the p21Waf1/Cip1 or the Bax
genes] pGL3-p21-Luciferase and pGL3-Bax-Luciferase, respectively). More
in details, pGL3-p21-Luciferase contains a 2.5 Kb stretch, corresponding to the
promoter sequence of the human p21Waf1/Cip1 gene digested with HindIII and
subcloned into the luciferase reporter vector pGL3-Basic-Vector and containing a tandem binding site for p53 (12); pGL3-Bax-Luciferase Bax luciferase
containing the proximal promoter region of the human bax gene fragment
-715/-317 from the Bax gene promoter subcloned into BglII–HindIII sites of
the pGL3-Basic-Vector (25,26). Transfected cells were treated with aspirin
(10 mM) for 24 h. Cells were harvested in 1 Reagent Lysis Buffer (Promega)
and the luciferase activity of the lysates was measured using the Luciferase
Assay System kit (Promega) following the manufacturer’s instructions.
Briefly, 50 mg of whole-cell lysates were mixed with 100 ml of reconstituted
Luciferase Assay Substrate, and the light emitted was measured with a Lumat
LB 9507 (Berthold, Vienna, Austria) tube luminometer. Relative units of
light were defined by the formula: relative units of light 5 specific units of
light/(control vector units basic vector units).
Aspirin induces G1 arrest and cell death in colon cells
It has been reported previously that aspirin causes cell cycle arrest and
induces apoptosis in a number of cell lines (5,29). In order to establish
whether the reduction in proliferation of colon cells was due to aspirininduced changes in cell cycle progression, HT29, HCT116 and
HCT116p53/ were cultured in the presence of 2.5, 5, or 10 mM
aspirin for 48 h and the cell cycle distribution was analyzed by flow
cytometry. Upon treatment with 5 mM aspirin (Figure 2A), HCT116
cells arrested in G1 almost completely (82% of the cell population)
within 48 h, whereas HCT116p53/ and HT29 cells showed a less
complete arrest (62 and 71%, respectively). High aspirin concentrations (10 mM) affected cell survival in HCT116 and HT29 but less
so in HCT116p53/ cells; the latter cell line arrested in both G1 and G2
(Figure 2A). The observed increase in the G1 population parallels the
changes in the MTT experiments with higher aspirin sensitivity displayed in HCT116 than in HT29 or HCT116p53/ cells. The strongest effect on G1 arrest was observed at 5 mM aspirin. As a control,
cells were treated with 100 lM sulindac sulfide (at its IC50 concentration, as assessed by MTT assay on HCT116 cells) for 48 h (Figure
2B), which has been described to reduce the proliferation rate of HT29 cells by inducing G1 arrest (30). We found that all three cell lines
responded to sulindac sulfide to a similar extent (G1 population:
HCT116 65%, HCT116p53/ 59% and HT29 68%). These results
suggest that p53 only partially controls the aspirin-mediated (but
not sulindac sulfide mediated) G1 arrest and at higher concentrations
the aspirin-induced cell death. However, cells lacking functional p53
were still able to activate checkpoint responses, which led to cell cycle
arrest more in G1 and G2 and, to a lower extent, to apoptosis.
Statistics
Proliferation inhibition by MTT was compared between different cell lines
using the Student’s T-test at a representative concentration. A P value below
0.05 was considered significant.
Aspirin activates p53 by phosphorylation at residue Ser15
The tumor suppressor p53 is known to play a key role in cell cycle
arrest as well as apoptosis in response to various stresses such as
Flow cytometry analysis
Cells treated with aspirin or sulindac sulfide for 48–72 h were harvested and
the cell cycle distribution was analyzed as described previously (23).
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Aspirin and cell cycle progression
DNA-damaging agents and anticancer drugs. Phosphorylation of p53
on Ser15 by the phosphatidylinositol-3-like kinases ATM and ATR
triggers post-translational modifications that contribute to p53 stabilization. Thus, we examined whether p53 protein levels and phosphorylation were altered by aspirin treatment in our system. HT29 and
HCT116 cells were exposed to 0, 2.5, 5 or 10 mM aspirin for 8 h.
Analysis of total cell lysates indicated a concentrationdependent increase in phosphorylation of p53 on Ser15 in both cell
lines but not of total protein levels (Figure 3A, top panels).
ATM is activated upon aspirin treatment in HCT116p53/ cells
One of the major phosphatidylinositol-3 kinases that target p53
N-terminal residue Ser15 for phosphorylation is ATM (31). HCT116
and HT29 cells were treated with 5 or 10 mM aspirin for 8 h, in the
presence or absence of caffeine, a known inhibitor of the ATM and ATR
pathways (32). Phosphorylation of p53 at Ser15 decreased in a caffeinedependent manner in HCT116 cells (Figure 3A, bottom panels) and in
HT29 cells (data not shown), suggesting a role for ATM or ATR in the
phosphorylation of p53 at Ser15. As we did not see activated ATR (i.e.
chromatin-bound ATR; data not shown), we investigated the activation
status of the ATM kinase by assessing its auto-phosphorylation at
Ser1981 upon exposure to aspirin. A time course experiment revealed
that ATM Ser1981 was phosphorylated within 2–4 h, with a peak at 8 h
in HCT116 cells (Figure 3B). The kinetic of ATM activation paralleled that of the phosphorylation of the histone c-H2AX at Ser139
(Figure 3B), a site targeted by ATM (19).
The checkpoint kinase Chk2 is activated upon aspirin treatment
As our data point to an activation of ATM by aspirin, we investigated
further ATM substrates such as Chk2. Chk2 kinase plays a central role
in modulating the cellular response to DNA damage, resulting in cell
cycle arrest, DNA repair or apoptosis depending on the severity of the
DNA damage and the cellular context. Chk2 is activated by phosphorylation on residue Thr68 by the kinase ATM (33). We treated
HCT116, HCT116p53/ and HT29 cells with 10 mM aspirin for 2,
4, 8 and 24 h. Although the kinase total levels were similar in all cell
lines, Chk2 phosphorylation at Thr68 was detectable in p53-null cells
as early as 2 h, whereas it was a much later event in the p53-wt cells
(Figure 3C). In this time course experiment, p53 phosphorylation at
Ser15 was visible within 8 h of aspirin treatment in HCT116 and
HT29 cells and correlated with ATM activation. As expected, no
p53 signal was detected in the HCT116p53/ cells. The timing of
phosphorylation of Chk2 and p53 is consistent with the activation of
the ATM pathway.
ATM inhibitor caffeine blocks the cellular responses induced by
aspirin
In order to further confirm the role of the checkpoint kinase ATM in
the aspirin-induced responses, we repeated the treatment of colon
cells HCT116 in the presence of caffeine, known to abrogate cellular
checkpoint responses dependent on ATM and ATR (7). HCT116 cells
were exposed to 5 or 10 mM aspirin for 48 h and 5 mM caffeine was
Fig. 1. Effect of aspirin on the proliferation of human colon cells. (A)
HCTl16 (white bar) and HT29 (gray bar) or (B) HCTl16 (white bar) and
HCTl16p53/ (gray bar) cells were plated at a density of 104 cells per well in
96-well microtiter plates. Twenty-four hours after seeding, the cells were
treated with aspirin (0–10 mM) for 72 h and analyzed by MTT assay. (C)
HCTl16p53/cells were transiently transfected with either pcDNA-p53
(black bars) or the empty plasmid pcDNA (gray bar); non-transfected
HCT116 cells (white bars) served as control. Twenty-four hours later, cells
were trypsinized and seeded at a density of 104 cells per well in 96-well
microtiter plates and subsequently treated with aspirin (0–10 mM) and
analyzed as in (A). The values are indicated as relative units (RU) normalized
to the untreated control and represent the mean of 4 separate measurements.
Aspirin caused a dose-dependent proliferation reduction that was more
pronounced in HCT116 cells than in HT29 or HCTl16p53/ cells; the reintroduction of the p53 protein in HCTl16p53/ cells increased sensitivity to
aspirin. (D) HCTl16p53/cells were transiently transfected with either the
pcDNA-p53 or the empty plasmid pcDNA and analyzed for the expression of
p53 48 h after transfection by western blotting. Cells transfected with
pcDNA-p53 showed over-expression of p53 when compared with the
endogenous levels present in HCT116 cells. Tubulin was used as loading
control.
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M.G.Luciani et al.
Fig. 2. Cell cycle distribution in aspirin- or sulindac sulfide-treated cells. HCTl16, HCTl16p53/ and HT29 cell lines were treated with 2.5, 5 and 10 mM aspirin
(A) or 100 lM sulindac sulfide (B) for 48 h. After staining with propidium iodide (PI), the cellular DNA content was visualized by flow cytometry and the cell
cycle distribution was analyzed. Cells with a sub-G1 DNA content (,G1) were considered to be apoptotic cells. HCT116 cells were more sensitive to aspirininduced G1 arrest than cells lacking a functional p53 protein; in contrast, all cell lines responded similarly to sulindac sulfide. The MTT assay for sulindac sulfide in
HCT116 cells is also shown (B). The gray line in the graph indicates its IC50 concentration (100 lM). The flow cytometry profiles are representative of at least 3
independent experiments.
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Aspirin and cell cycle progression
added either at the same time as aspirin or 24 h later (Figure 3D).
Analysis of cell cycle progression revealed that caffeine was able to
prevent the activation of the aspirin-dependent G1 arrest and apoptosis; moreover, this effect was time dependent, as exposure to caffeine
for 24 h only partially reversed both responses. Taken together, all
these results indicate that the ATM checkpoint kinase mediates the
effect of aspirin inhibition of proliferation in colon cells.
Aspirin-induced activation of caspase 7 is an early event
The cleavage and activation of caspase 7 is a well-established marker
for the onset of apoptosis (34). When we tested whether the caspase
cascade was activated in colorectal cell lines, we found that aspirin
increased cleavage of caspase 7 (Figure 3C). Activation of p53 is
known to mediate apoptosis, which has been observed upon treatment
with aspirin or NSAIDs (5). Caspase 7 cleavage was more pronounced
in the p53-wt cells and happened at an earlier time point (2 h). In the
p53-mutant HT29 cells, the signal of caspase 7 cleavage was delayed
but strong after 24 h, consistent with the pronounced cell death described earlier (Figure 2A), whereas in the HCT116p53/ cells, caspase 7 cleavage was much weaker (also consistent with the lower
extent of cell death shown in Figure 2A). These data suggest that
aspirin induces apoptosis mostly in cells expressing either wild-type
or mutant p53. However, a p53-independent mechanism must be responsible for the cleavage of caspase 7 (although weak) and cell death
in p53-null cells.
Expression of the CDK inhibitor p21Waf1/Cip1 is essential for the
aspirin-induced G1 arrest in colorectal cells
It is well established that p53 mediates cell cycle arrest in the G1
phase through transcriptional activation of p21Waf1/Cip1, an inhibitor
of the CDKs 2, 3, 4 and 6. In HCT116 cells, an increase in p21Waf1/Cip1
levels was observed after 16 h treatment with aspirin (Figure 3C).
However, the levels of p21Waf1/Cip1 also increased in p53-null and
mutant cells, which correlated with increased phosphorylation levels
of Chk2 (Figures 3C), indicating the presence of a p53-independent
mechanism for p21Waf1/Cip1 up-regulation in these cells. A regulation
of p21 levels by aspirin has been reported previously in HT29 cells,
which express the transcriptionally inactive p53R273H mutant (35). In
order to further investigate the role of p21Waf1/Cip1, we extended our
study to HCT116p21/ cells, in which the expression of the p21Waf1/Cip1
gene has been disrupted [Figure S1, supplementary data are available
at Carcinogenesis online; (22)]. HCT116p21/ cells were even more
resistant to aspirin than HCT116p53/ cells (e.g. at 2.5 mM aspirin
76.8 ± 10.3 versus 47.6 ± 2.1% of control; P , 0.0001; Figure 4A).
Moreover, 48 h of treatment with aspirin failed to induce a G1 arrest
(Figure 4C), which was clearly visible in the parental cell line
HCT116 (Figure 4B). Instead, even low concentrations of aspirin
(above 1.25 mM) induced cell death, suggesting that this apoptotic
response is not dependent on p21Waf1/Cip1 but also that the disruption
of p21Waf1/Cip1 could somehow sensitize the cell to apoptotic stimuli
(36). In order to quantify the effect of aspirin on p21Waf1/Cip1 expression, we transfected HCT116, HCT116p53/, HCT116p21/ and
HT29 cells with a luciferase reporter plasmid bearing the p21Waf1/Cip1
minimal promoter upstream of the Luciferase gene. Upon aspirin
treatment, all cell lines showed an increased expression of p21Waf1/Cip1driven luciferase activity (Figure 4D), consistent with our western blot
findings (Figure 3C). In an analogous experiment with a luciferase
reporter plasmid containing the Bax promoter, only HCT116p53/
failed to induce Bax-driven luciferase expression (Figure 4D). Furthermore, when p53 was reintroduced into the HCT116p53/ cells
via transient transfection, they re-gained the ability to up-regulate the
expression of Bax upon treatment with aspirin (Figure 3D), suggesting
that the induction of Bax, the major mediator of apoptosis upon DNA
damage, is dependent on p53 expression (37).
Aspirin-induced G1 arrest is reversible
Activation and phosphorylation of the checkpoint kinases ATM and
Chk2, as well as p53 phosphorylation at Ser15, are normally associ-
ated with cellular responses to DNA damage and/or DSBs. Moreover,
the appearance of foci of c-H2AX phosphorylation is characteristic of
DNA damage and repair. In order to verify that aspirin did not cause
permanent damage to cells, we exposed HCT116 cells to 5 or 10 mM
aspirin for 48 h followed by culturing these cells under normal medium conditions for further 48 h (Figure 5). The G1 arrest and other
cellular changes (which might mediate a G1/S checkpoint response)
were fully reversible, as the profiles of the cell samples were very
similar to those of untreated cells. In addition, after treatment with
10 mM aspirin, the remaining viable cells seemed to regain a normal
proliferation profile. This suggests that aspirin does not produce permanent cell damage.
Discussion
There is growing experimental and clinical evidence indicating that
aspirin has cancer-preventive activity, in particular, in the colon (1).
A possible mechanism for the antitumor properties of aspirin has been
ascribed to its direct inhibition of COX-2 in colorectal cells (38).
However, the anti-proliferative activity of aspirin does not correlate
exclusively with its COX-2 inhibitory activity, as aspirin can induce
apoptosis in cells lacking COX-2 expression [see Figure S1, supplementary data are available at Carcinogenesis online and (39)]. Although we have not tested the inhibition of prostaglandin production
in our system, we do not believe that the observed effects may depend
on the regulation of the prostaglandin pathway alone, as the latter is
likely to be similarly regulated in the nearly isogenic cell lines
HCT116, HCT116p53/ and HCT116p21/, all negative for COX-2
(Figure S1, supplementary data are available at Carcinogenesis
online), whereas these cell lines show distinctively different responses
to aspirin.
Predisposition to cancer and radiosensitivity observed in Ataxia
Telengectasia patients has been linked to chromosomal instability,
abnormalities in genetic recombination and defective signaling to
programmed cell death and several cell cycle checkpoints activated
by DNA damage. ATM mutations (generally null alleles that truncate
or destabilize the protein) are involved in the development of sporadic
human cancers such as leukemia (40); loss of heterozygosity at the
ATM locus was also found in about 30% of colorectal carcinomas
(41). These observations predicted that the ATM gene encodes a protein, which plays a crucial role in sensing DNA damage and transducing signals that promote apoptosis (42). Furthermore, it was
reported that ATM might have a role in activating defence mechanisms against oxidative stress (43). In a recently published study, the
mechanisms by which the aspirin-related compound nitric oxide (NO)releasing aspirin induced apoptosis in the human B-lymphoblastoid
TK6 cell line were investigated. The authors observed that treatment
with NO-aspirin led to DNA damage, histone H2AX phosphorylation
on Ser 139 and ATM phosphorylation on Ser 1981, effects that were
dependent on the NO-moiety of the compound. In fact, these responses were attenuated by the reactive oxygen species scavenger
N-acetyl-L-cystein, suggesting that the DNA damage induced by
NO-aspirin is caused by oxidative stress. In our system, aspirin did
not affect the production of reactive oxygen species by activated polymorphonuclear neutrophils (Supplementary Figure S4, supplementary
data are available at Carcinogenesis online), as measured by a lucigenin-based method, indicating that oxidative stress is not the mediator
of the observed response to aspirin. We reported the activation of the
ATM-dependent checkpoint independently of oxidative stress and of
aspirin anti-inflammatory properties. Our evidence for ATM being
involved in the checkpoint response mediated by aspirin includes
auto-phosphorylation of ATM and of its targets c-H2AX, p53 and
Chk2 (Figure 3A–C) and the ability of the ATM inhibitor caffeine
to revert the cell cycle arrest and apoptosis (Figure 3D).
Oncogenes and tumor suppressor genes play essential roles in colorectal carcinogenesis. In cancer cells defective for p53, the tumor
suppressor is no longer able to control cell proliferation and is
inefficient in preventing the emergence of genetically unstable
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Fig. 3. Aspirin induces activation of components of checkpoints and apoptotic pathways. (A) Aspirin induces phosphorylation of p53 in colon cancer cells.
HCT116 and HT29 cells were treated with 0, 2.5, 5 and 10 mM aspirin for 8 h. The upper western blots show the total expression levels of p53 (upper panel) and
the phosphorylation of p53 at Ser15 (lower panel) in cell lysates of treated cells. A dose-dependent phosphorylation of p53 is visible in both cell lines. Lower
western blot: phosphorylation of p53 at Ser15 is reduced by caffeine. HCTl16 cells were treated with either 5 or 10 mM aspirin for 8 h in the presence or absence of
caffeine (5 mM) and cell lysates were analyzed for total levels of p53 (upper panel) and phosphorylation at Ser15 (lower panel). Caffeine treatment reduces p53
phosphorylation by aspirin to levels similar to the control. (B) ATM is phosphorylated upon aspirin treatment. HCTl16 and HCTl16p53/ cells were treated with
either 10 mM aspirin (ASA) for 8 h and cell lysates analyzed by immunoblotting for phosphorylation of ATM at residue Ser1981 (upper panel). Phosphorylation of
ATM at Ser1981 is induced by aspirin in both cell lines within 8 h. Phosphorylation of p53 at Ser15 (middle panel) and expression of p21Waf1/Cip1 (lower panel)
were also analyzed. The DNA polymerase-a inhibitor aphidicolin (40 lM) was used as a positive control for p53 Ser15 phosphorylation. The different levels of
basal expression of p53 phosphorylation between the experiments in 3B and 3C are due to different amounts of total protein loaded in each lane (50 versus 150 lg).
(C) ATM activation peaks at 8 h and correlates with c-H2AX Ser139 phosphorylation. HCT116 cells were treated with 10 mM aspirin for 2, 4, 8 or 24 h and total
cell lysates were tested for ATM and c-H2AX phosphorylation. ATM phosphorylation at Ser1981 is visible within 2 h and reaches its highest level at 8 h.
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Aspirin and cell cycle progression
Fig. 4. p21Waf1/Cip1 is required for the aspirin-induced G1 arrest. (A) HCTl16 (white bars), HCT116p53/ (black bars) and HCT116p21/ (black bars) cells were
analyzed for proliferation as described in Figure 1. HCT116p21/ cells were most resistant to aspirin. Cell cycle distribution of HCT116p21/ cells in the
presence of aspirin. HCTl16 (B) and HCT116p21/ (C) cell lines were treated with 0.6–10 mM aspirin for 48 h and analyzed as described in Figure 2. In
HCT116p21/ cells, aspirin does not induce a G1 arrest but cell death. (D) Aspirin induces p21Waf1/Cip1 expression in a p53-independent manner. Upper panels:
HCTl16, HCTl16p53/, HCTl16p21/ and HT29 cells were transfected with 0.5 lg of pGL3-Basic-Vector, pGL3-Control-Vector, pGL3-p21-Vector or pGL3Bax-Vector. Lower panel: HCT116p53/ cells were transfected 0.5 lg of pGL3-Basic-Vector, pGL3-Control-Vector or pGL3-Bax-Vector in combination with
either pcDNA empty vector or pcDNA-p53. Twenty-four hours after transfection, cells were treated with 10 mM aspirin for an additional 24 h; then cells were
lysed and the luciferase activity was measured (expressed as relative units of light 5 RUL). The graphs show RUL of untreated (white bars) and aspirin-treated
(gray) cells (R 5 ratio of aspirin treated/untreated). Aspirin increases the p21-driven expression of luciferase in all cell lines. In contrast, Bax-driven luciferase
expression is only increased in cells expressing p53.
Phosphorylation of c-H2AX at Ser139 follows a similar kinetics. Total levels of ATM and c-H2AX, as well as a-tubulin, are shown as loading controls. (D) Chk2 is
phosphorylated and caspase 7 cleaved in aspirin-treated cells. HCTl16, HCTl16p53/ and HT29 cells were cultured in the presence of 10 mM aspirin (ASA) for 2,
4, 8, 16 or 24 h and the total cell lysates analyzed by immunoblotting for phosphorylation of Chk2 at residue Thr68 and total levels; phosphorylation of p53 at
residue Ser15 and total levels, as well as for expression of p21Waf1/Cip1 and cleavage of caspase 7. Tubulin was used as a loading control. In p53-expressing cells,
phosphorylation of p53 at Ser15 was visible within 8 h after treatment with 10 mM aspirin and correlated with increased p21Waf1/Cip1 expression. The latter was
also observed in HCTl16p53/ cells. Chk2 was phosphorylated at Thr68 in all cell lines; phosphorylation was highest in HCTl16p53/ cells, and correlated with
increased levels of p21Waf1/Cip1. Caspase 7 was cleaved upon aspirin treatment in all cells; its activation was an early event in HCT116 cells (2 h) but not in HT29
cells.
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M.G.Luciani et al.
Fig. 4. Continued
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Aspirin and cell cycle progression
Fig. 5. Aspirin-induced G1 arrest is reversible in colon cells. (A) HCTl16 cells were treated with 5 or 10 mM aspirin for 48 h (upper panels). Viable cells were
returned to normal culture medium for an additional 48 h (lower panel). Cells were harvested and the DNA content analyzed upon propidium iodide staining (PI).
The G1 arrest observed with 5 mM aspirin was fully reversible upon aspirin wash out. (B) HCTl16 cells were treated with 5 or 10 mM aspirin for 48 h either alone
(upper panels) or in combination with 5 mM caffeine for 24 h (middle panels) or 48 h (lower panels). The presence of caffeine reversed the G1 arrest and the
apoptotic response induced by aspirin in a time-dependent manner.
cell populations. The most common changes of p53 in cancer tissues are missense mutations and loss of heterozygosity, leading to a
Ôloss-of-functionÕ phenotype. Such alterations are found in many human cancers, including colon carcinomas (incidence of 70–75% of
cases) (44). Phosphorylation at different residues in p53 has been
shown to occur after cells were exposed to DNA-damaging agents.
Upon cellular stress, p53 is phosphorylated by kinases targeting its Cterminal and N-terminal domains, which results in the activation of its
transcriptional activity (24,45). Serine 15 is a known target of ATM
and becomes phosphorylated after DNA damage (46). In our study,
p53 phosphorylation at Ser15 was increased following aspirin treatment and was sensitive to the ATM and ATR inhibitor caffeine (Figure
3A). Caffeine was also able to reverse the cellular responses of G1
arrest and apoptosis induced by aspirin in a time-dependent manner
(Figure 3D). Moreover, we detected ATM auto-phosphorylation, as
well as phosphorylation of c-H2AX and Chk2 at ATM-targeted sites
(Figure 3B and C). Phosphorylation of c-H2AX by ATM facilitates
the formation of foci to which proteins known as mediators of the
DNA damage checkpoint, such as MDC1 and BRCA1, are recruited
(19). The assembly of these factors near a DSB coincides with checkpoint activation and recruitment of repair proteins. Therefore, the
presence of c-H2AX phosphorylation suggests that aspirin generates
signaling resembling that of DSB-induced checkpoint. However, the
exact nature of the molecular signal that activates this checkpoint
remains to be determined.
Our results show that aspirin induces a G1 cell cycle arrest. An
aspirin-dependent G1 arrest had been observed before in several tumor
cell lines (5,29). A recent study (47) investigated the effects of aspirin
on the NF-jB signaling in HCT116-derived cell lines, with regards to
NF-jB translocation and induction of apoptosis. The authors found no
effect of either p53 or hMLH1 in the inhibition of the NF-jB pathway
by aspirin. A close analysis of some of their findings, though, also
shows differences in the cell viability and response to apoptosis between HCT116 and HCT116p53/ cells, which match our results. We
therefore suggest that p53 has only a minor role in the cell cycle
response to aspirin, as cells not expressing p53 still undergo a good
G1 arrest. It has also been reported that the tumor suppressor p53 may
up-regulate COX-2 expression and that COX-2 in turn inhibits p53dependent transcription (48). However, as we showed, and others
reported (49) that HCT116 cells do not express COX-2, this is unlikely to be the molecular mechanism behind the response to aspirin.
In our study, we pinpoint the CDK inhibitor p21Waf1/Cip1 as the
major mediator for the aspirin-dependent G1/S checkpoint. A recent
microarray-based study by Hardwick et al. (35) reported p21Waf1/Cip1
to be up-regulated, at both gene and protein levels, in HT29 cells
treated with 5 mM aspirin. Consistently with these findings,
p21Waf1/Cip1 were also raised in our system in all cells tested, including HCT116p53/ and HT29 cells (Figure 3C; Figure S1, supplementary data are available at Carcinogenesis online). In HT29 cells, p53 is
transcriptionally inactive, bearing a mutation (R273H) within its
DNA-binding domain, whereas HCT116p53/ do not express p53;
therefore, the increase in the p21Waf1/Cip1 expression upon aspirin
treatment is likely to be p53 independent.
The checkpoint kinase Chk2 has also been described to induce
p21Waf1/Cip1-dependent senescence (50). Some other p53-independent
mechanisms of increasing p21Waf1/Cip1 expression have been reported
(51). Among these, the tumor suppressor kinase LKB1, which is often
mutated in sporadic cancers (such as CRC), has been recently linked
to p21Waf1/Cip1 expression (52). Germline mutations of LKB1 lead to
Peutz–Jeghers syndrome, which is characterized by gastrointestinal
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M.G.Luciani et al.
Fig. 6. Model of the aspirin-induced checkpoint activation. In this model,
solid arrows correspond to observations derived directly from our results,
dashed arrows indicate possible other interactions and the gray arrows
indicate interactions that may also take place as reported previously from
literature. Aspirin may synergize with checkpoint sensor proteins upstream
of an ATM-dependent cellular pathway. ATM, activated by autophosphorylation and consequent disruption of the homodimer structure,
phosphorylates p53 on Ser15. ATM-dependent phosphorylation of p53
results in protein stabilization and (together with additional phosphorylation
by other kinases, such as CDK2–cyclin A) in transcriptional activation of
downstream target genes, such as p21Waf1/Cip1 (a major mediator of the G1
arrest) and the pro-apoptotic protein Bax. Our data also suggest that aspirin
induces expression of p21Waf1/Cip1 in a p53-independent manner through an
(unknown) additional factor. In addition, ATM phosphorylates the
checkpoint kinase Chk2 on several sites, including Thr68. Chk2 is primarily
involved in the phosphorylation of p53 at Ser20 and in the further
stabilization of the tumor suppressor (via phosphorylation of MDM2).
Recent reports suggest that Chk2 may interact with p21Waf1/Cip1 to mediate a
p53-independent apoptotic response and cellular senescence in human cells (50).
polyps and cancer in different organs and to loss of LKB1 kinase
activity (53). It would be interesting to investigate whether aspirin
has any effect on the activation of the LKB1 kinase pathway and if it is
useful in patients with Peutz–Jeghers syndrome.
Interestingly, we observed cell death (Figure 2A), caspase 7 cleavage (Figure 3C) and Bax-driven luciferase expression (Figure 4D) not
only in wild-type but also in cells expressing p53R273H (but not in
p53-null cells), suggesting that the mutant p53 is able to induce Bax
expression, activation of the caspase cleavage cascade and thereby
apoptosis. HT29 cells have been described previously as being sensitive to aspirin- or other drug-induced apoptosis (54). It was shown that
elevated levels of p53R273H, although considered to be transcriptionally inactive, rendered transformed human cells susceptible to apoptosis through Bax (55). Our luciferase reporter assay indicates that
p53R273H is able to bind to the Bax promoter, which might suggest that
aspirin maintains chemopreventive activities in tumors in which mutations of p53 have occurred. We could speculate that this mechanism
helps preventing clonal expansion of p53-mutated cells upon aspirin
treatment.
Upon treatment with 10 mM aspirin, HCT116p53/ cells accumulate in G1 and G2, indicating that both checkpoints have been activated
(Figure 2A, middle panels). In a previous study with p53-deficient
cells, a bypass of the G1/S checkpoint and an increase in G2/M arrest
was described in response to DNA damage (56). Chk2 kinase, which
mediates the cellular responses to DNA damage downstream of ATM
(33), is known to induce arrest in the G2/M phase of the cell cycle by
inhibiting the dual phosphatase Cdc25C and thus blocking activation
2216
of the CDC2–cyclin B complex and entry into mitosis (32). In
HCT116p53/ cells, a particularly strong phosphorylation of Chk2
was also observed (Figure 3C). These data lead us to hypothesize that,
at high concentrations of aspirin, Chk2-dependent G2/M checkpoint
pathways are activated in p53-deficient cells along a weak apoptotic
response.
Our results taken together suggest a novel ATM-dependent mechanism of action for aspirin in the chemoprevention of colon cancer.
Aspirin is mainly operating through the p21Waf1/Cip1 pathway for its
anti-proliferative activity in a p53-independent manner. However,
cells not expressing p53 are more resistant to aspirin-induced cell
death suggesting that p53 has an important role in the activation of
the apoptotic response in colon cells. Downstream effects of the ATM
activation induce cleavage of caspase 7 and up-regulation of p21Waf1/Cip1
and Bax; the expression of p21Waf1/Cip1 upon aspirin treatment is
transcriptionally regulated in the absence of p53 and may be dependent upon additional factors (Figure 6). It has been suggested that
ATM is recruited to the site of DNA damage. ATM can directly bind
to and phosphorylate proteins involved in DNA repair, such as c-Abl,
Brca1, Nbs1 and replication protein A (57). We speculate that, by
activating the G1/S checkpoint, aspirin increases the opportunity of
colorectal cells to repair DNA damage before replication or to induce
apoptosis both of which may contribute to maintaining the integrity of
genomic DNA. Sensor protein complexes scan the DNA for abnormalities and translate these stimuli into activating signals for downstream target proteins, such as the ATM kinase. However, despite the
numerous studies on molecular components of checkpoints, both the
identities of these sensors and their mechanisms of action are still
unclear. It seems possible that aspirin interferes with such sensors
upstream of ATM. The identification of additional aspirin targets
(besides COX-2) will help in the understanding of its antitumor effects and in the design of novel chemopreventive agents.
Aspirin is rapidly hydrolyzed to salicylate and other salicylic metabolites both in vivo (58) and in vitro (59). The clinical relevance of
our data is strengthened by the fact that these concentrations of aspirin
correspond to salicylate levels measured in the plasma of human
patients, as well as to the therapeutic concentrations used in the treatment of arthritis (60). Comparisons between concentrations used on
cell cultures and plasma levels, however, are somewhat artificial,
because of the inability to accurately mimic in vivo metabolism and
tissue concentration of aspirin in epithelial or tumor cells. In addition,
the duration of drug exposure in cell culture is minimal when compared with long-term use for the reason of chemoprevention. In our
study, the effects of aspirin on apoptosis and cell cycle were greatest
when the cells were treated with a single dose of 5–10 mM aspirin for
48 h. A dose-dependent response in G1 arrest was already visible at
lower concentrations in HCT116 cells (1.25–2.5 mM). Therefore,
although the concentrations of aspirin used in our study may seem
rather high, they are within the range that has been used in culture
previously (1–20 mM) (4,5,61), and the effects observed may accurately reflect a biomechanism of repetitive dosing for long-term
chemoprevention.
Supplementary material
Supplementary figures S1–S4 can be found at http://carcin.
oxfordjournals.org/
Acknowledgements
We would like to thank Prof. Ted Hupp (University of Edinburgh, UK) for the
kind gift of the HCT116p53/ cells, HCT116p21/ cells and the Luciferase
plasmids, pGL3-p21-Luciferase and pGL3-Bax-Luciferase, Ms Cornelia Lichtenberger (Medical University of Vienna, Vienna) for her valuable help with
the flow cytometry experiments and Dr James Hutchins (Research Institute for
Molecular Pathology, Vienna) and Prof. Thurnher (Medical University of Vienna,
Vienna) for the COX-1 and COX-2 antibodies. This work was supported by the
Austrian Science Fund grants M-874-B14 (MGL) and P18270 (CG).
Aspirin and cell cycle progression
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Received December 18, 2006; revised March 19, 2007; accepted April 17,
2007
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