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5-Aza-2ⴕ-deoxycytidine Activates the p53/p21
Waf1/Cip1
Pathway to
Inhibit Cell Proliferation*
Received for publication, October 24, 2003, and in revised form, January 12, 2004
Published, JBC Papers in Press, January 13, 2004, DOI 10.1074/jbc.M311703200
Wei-Guo Zhu‡§¶, Theresa Hileman储, Yang Ke§, Peichang Wang‡, Shaoli Lu‡, Wenrui Duan储,
Zunyan Dai**, Tanjun Tong‡, Miguel A. Villalona-Calero储, Christoph Plass**,
and Gregory A. Otterson储‡‡
From the ‡Department of Biochemistry and Molecular Biology and the §Cancer Research Center, Peking University
Health Science Center, 38 Xueyuan Road, Beijing 100083, China, the 储Division of Hematology/Oncology, Department of
Internal Medicine, and the **Division of Human Cancer Genetics, Comprehensive Cancer Center, The Ohio State
University, Columbus, Ohio 43210
In addition to its demethylating function, 5-aza-2ⴕ-de-
oxycytidine (5-aza-CdR) also plays an important role in
inducing cell cycle arrest, differentiation, and cell
death. However, the mechanism by which 5-aza-CdR in-
duces antineoplastic activity is not clear. In this study,
we found that 5-aza-CdR at limited concentrations
(0.01–5
M) induces inhibition of cell proliferation as
well as increased p53/p21
Waf1/Cip1
expression in A549
cells (wild-type p53) but not in H1299 (p53-null) and
H719 cells (p53 mutant). The p53-dependent p21
Waf1/Cip1
expression induced by 5-aza-CdR was not seen in A549
cells transfected with the wild-type human papilloma
virus type-16 E6 gene that induces p53 degradation. Fur-
thermore, deletion analysis and site-directed mutagen-
esis of the p21 promoter reveals that 5-aza-CdR induces
p21
Waf1/Cip1
expression through two p53 binding sites in
the p21 promoter. Finally, 5-aza-CdR-induced p21
Waf1/
Cip1
expression was dependent on DNA damage but not
on DNA demethylation as demonstrated by comet assay
and bisulfite sequencing, respectively. Our data provide
useful clues for judging the therapeutic efficacy of 5-aza-
CdR in the treatment of human cancer cells.
As demethylating agents, 5-aza-cytidine and 5-aza-2⬘-deoxy-
cytidine (5-aza-CdR)
1
have been extensively used for epigenetic
research (1– 4). Both demethylating agents are incorporated
into DNA where they bind DNA methyltransferase (DNMT) in
an irreversible, covalent manner, thus sequestering the en-
zyme and preventing maintenance of the methylation state
(5–7). Consequently, silenced genes induced by hypermethyla-
tion are re-expressed after treatment with these demethylating
agents.
Originally, 5-aza-cytidine and 5-aza-CdR were developed as
anticancer agents (5, 8) and have been shown to have signifi-
cant cytotoxic and antineoplastic activities in many experimen-
tal tumors (9 –12). 5-Aza-CdR is reported to be noncarcinogenic
and incorporates into DNA but not RNA or protein (13, 14).
5-Aza-CdR has been found empirically to have more potent
therapeutic effects than 5-aza-cytidine in cell culture and ani-
mal models of human cancers.
However, 5-aza-CdR-induced cytotoxicity may not be linked
to its demethylating function (3, 15–17). In addition, the ther-
apeutic effects of 5-aza-CdR in the treatment of different hu-
man cancer cells are conflicting. 5-Aza-CdR appears to be ben-
eficial in the treatment of human leukemias (9, 18, 19),
myelodysplastic syndromes (20, 21), and hemoglobinopathies
(22, 23). On the other hand, there has been less positive expe-
rience in the effectiveness of 5-aza-CdR for the treatment of
human solid tumors (10, 24). Therefore, it is possible that one
or more critical factors may be involved in regulating the cel-
lular response to 5-aza-CdR treatment that vary in different
cell types.
p53 is a very important tumor suppressor gene and is re-
ported to be abnormal in more than 50% of human cancers (25).
Chemotherapeutic agents frequently act through the mecha-
nism of DNA damage, and p53 plays an important role in the
induction of cell cycle arrest and apoptosis in response to DNA
damage (26). 5-Aza-CdR has also shown anticancer activity
that may be related to its ability to induce DNA damage (15,
27). Based on the scenario mentioned above, it is hypothesized
that 5-aza-CdR may induce DNA damage, thereby activating
p53, which in turn increases p21
Waf1/Cip1
expression, leading to
the inhibition of cell proliferation.
To confirm the role of p53 in the 5-aza-CdR-induced inhibi-
tion of cell proliferation, human lung cancer cells with different
p53 status were selected as the targets for this study. As an
important downstream target of p53 activation, p21
Waf1/Cip1
plays a critical role in inhibiting cell proliferation; therefore,
p21
Waf1/Cip1
expression upon treatment of 5-aza-CdR in cells
with different p53 status was given special attention in this
study. Present data indicated that 5-aza-CdR is a strong DNA-
damaging agent, and 5-aza-CdR induces inhibition of cell pro-
liferation by activating the p53/p21 pathway.
EXPERIMENTAL PROCEDURES
Cells and Treatments—Human lung cancer cell lines A549, H1299,
and H719 were grown in RPMI 1640 supplemented with 10% fetal
bovine serum (heat-inactivated at 56 °C for 45 min) and penicillin/
streptomycin, in a humidified, 5% CO
2
atmosphere and 37 °C incubator.
These cells were treated with 5-aza-CdR (0.01–20
M; Sigma) for up to
72 h. Fresh medium containing 5-aza-CdR was added every 24 h. The
treated cells then were washed with phosphate-buffered saline, placed
* This work was supported by 973 Plan Grant 2002CB713701 and
National Natural and Scientific Foundation Grant 30371613, by a Pe-
king University 985 Plan grant (to W.-G. Z.), by a Translational Re-
search Grant from the Valvano Foundation (to G. A. O.), and by Grant
P30CA16058 from the NCI, National Institutes of Health (Bethesda,
MD). The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
¶To whom correspondence may be addressed. E-mail: zhuweiguo@
bjmu.edu.cn.
‡‡ To whom correspondence may be addressed. Tel.: 614-293-6786;
Fax: 614-293-7529; E-mail: otterson-1@medctr.osu.edu.
1
The abbreviations used are: 5-aza-CdR, 5-aza-2⬘-deoxycytidine;
DNMT, DNA methyltransferase; RLU, relative luciferase activity;
HPV, human papilloma virus; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; CG, cytosine-guanine dinucleotide.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 15, Issue of April 9, pp. 15161–15166, 2004
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org 15161
by guest, on December 14, 2010www.jbc.orgDownloaded from
in drug-free medium, and harvested at 24 h after incubation at 37 °C.
Western Blotting—Protein expression was detected by Western blot-
ting as previously described with minor modifications (12, 16). Briefly,
the cells were harvested with a scraper and then washed with cold
phosphate-buffered saline once. The cells were then lysed in lysis buffer
(50 mMTris-HCl, 250 mMNaCl, 5 mMEDTA, 50 mMNaF, 0.15% Igepal
CA-630, and 1.5 mMphenylmethylsulfonyl fluoride). Equal amounts of
proteins (100 –150
g) were size-fractionated on 9 –15% SDS-PAGE.
The antibodies used are anti-p21
Waf1/Cip1
(F-5; Santa Cruz; 1
g/ml),
anti-p53 (DO-1; Oncogene Research Products; 0.5
g/ml), and
␣
-tubulin
(Oncogene Research Products; 0.3
g/ml).
Generation of Stable Clone and Transfection—Wild-type human pap-
illoma virus type-16 E6 gene (HPV E6) was a gift from Dr. H. Ding
(Department of Radiology, Ohio State University) (28). The HPV E6
gene was inserted into pCMV-neo, and the pCMV-neo-E6 was trans-
fected into A549 cells by using a transfection kit (Qiagen) according to
the manufacturer’s instructions. The stable clone of A549-pCMV-
neo-E6 (A549-E6) was maintained in medium containing G418 at
500
g/ml.
Methylation-specific PCR and Methylation Detection—DNA was ex-
tracted and then treated with bisulfite as previously described with
minor modifications (29). Briefly, genomic DNA (1
g) in a volume of 50
l was denatured by NaOH (final concentration, 0.275 M) for 10 min at
42 °C. The denatured DNA was then treated with 10
lof10mM
hydroquinone and 520
lof3Msodium bisulfite at 50 °C overnight. The
primers for p21 were as follows: forward primer, 5⬘-GGG AGG AGG
GAA GTG TTT TT-3⬘, and reverse primer, 5⬘-ACA ACT ACT CAC ACC
TCA ACT-3⬘. The PCR conditions were initiated with a denaturing step
of 95 °C for 10 min, followed by 36 cycles of 96 °C for 30 s, 53 °C for 20 s,
and 72 °C for 20 s and were concluded with 72 °C for 7 min. The PCR
products were purified with a purification kit (Qiaquick) and then
incubated with HhaI (50 °C) for 2 h and TaqI at 65 °C for 2 h, respec-
tively. Digested DNA was then size-fractionated via polyacrylamide gel
electrophoresis to detect the methylation status.
Bisulfite Sequencing—DNA was treated with bisulfite and purified
for PCR as described above. The PCR products were gel-extracted
(Qiagen) and ligated into a plasmid vector, pCR2.1-TOPO, using the TA
cloning system (Invitrogen). Plasmid-transformed bacteria TOP10 F⬘
were cultured overnight, and the plasmid DNA was isolated (Qiagen).
At least 10 separate clones were chosen for sequence analysis.
Transient Transfection and Measurement of Relative Luciferase Ac-
tivity—Vectors used for transfection include pWWP-Luc and pWP101-
Luc (30, 31). The human wild-type p21 promoter luciferase fusion
plasmid, pWWP-Luc, was made from a 2.4-kb genomic fragment of p21
promoter containing the transcriptional start site and two p53 binding
sites and then subcloned into the luciferase reporter vector, pGL-3Ba-
sic. pWP101-Luc does not contain any p53 binding site. A549 cells
transfected with the pWWP-Luc or the pWP101-Luc were treated with
5-aza-CdR (0.5–5
M, 24 h) and then harvested to analyze the relative
luciferase activity.
Site-directed Mutagenesis—Mutant p21 promoter constructs were
generated using a site-directed mutagenesis kit (QuikChange; Strat-
agene, La Jolla, CA). pWWP-Luc (containing two p53 binding sites) was
used as the mutagenesis template. The p53 recognition elements con-
sist of four tandem PuPuPuC (A/T) pentamers (32). The first p53 bind-
ing site, GAACA (33) (⫺2234 to ⫺2230 relative to the translational start
site) was replaced with GAAAC, and the second binding site, AGACT
(33) (⫺1344 to ⫺1340 relative to the translational start site) was re-
placed with AGAAT following the manufacturer’s directions.
Comet Assay for Detecting DNA Strand Breaks—The comet assay,
also called the single-cell gel electrophoresis, was performed as de-
scribed previously (34). In brief, fully frosted microscopic slides were
covered with 110
l of 0.5% normal melting agarose at 60 °C. The slides
were immediately covered with a coverslip and then kept at 4 °C for 15
min to allow the agarose to solidify. About 10
5
cells of 5-aza-CdR treated
or untreated cells in 40
l of phosphate-buffered saline were mixed with
equal amount (40
l) of 1% lower melting agarose to form a cell sus-
pension. After gently removing the coverslip, the cell suspension was
pipetted onto the first agarose layer, spread using a coverslip, and
maintained at 4 °C for 15 min to allow it to solidify. After removal of the
coverslips, the slides were immersed in fresh prepared cold lysing
solution (2.5 MNaCl, 100 mMNa
2
EDTA, 10 mm Tris, pH 10.0, 1%
sodium sarcosinate) with 1% Triton X-100 for 40 min at 4 °C. The slides
were then placed in a horizontal gel electrophoresis tank filled with
fresh electrophoresis solution (1 mMNa
2
EDTA, 300 mMNaOH, pH
13.0) for 10 min. The slides were then placed in Tris buffer (0.4 MTris,
pH 7.5) for 15 min twice (neutralizing the excess alkali) after electro-
phoresis at 4 °C. The slides were then stained with 75
l of propidium
iodide (5
g/ml) for 30 min.
The slides were examined at 600⫻magnification, and the pictures
were taken under a fluorescence microscope (TCS; Leica, Mannheim,
Germany). To score the percentage of DNA in the tail, the image
FIG.1.5-Aza-CdR-induced cell proliferation inhibition is asso-
ciated with p21
Waf1/Cip1
expression. Cell viability was measured
with MTT when A549, H1299, and H719 cells were treated with 5-aza-
CdR at 0 –20
Mfor 72 h (A) and at 1
Mfor different intervals (B). C,
representative Western blots show changes in p21
Waf1/Cip1
expression in
A549 or H1299 cells after treatment with 5-aza-CdR at different con-
centrations.
␣
-Tubulin is presented as a loading control.
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analysis system was used (Q550CW; Leica). The percentage of comet
tail area (DNA tail area/total DNA area) and the comet tail length (from
the center of the DNA head to the end of the DNA tail) were analyzed
in 50 cells for one slide.
RESULTS
Dose-dependent Inhibition of Cell Proliferation by 5-Aza-
CdR—In this study, human lung cancer cell lines A549 (wild-
type p53), H1299 (p53-null), and H719 (mutant p53) were
treated with 5-aza-CdR at different concentrations for 72 h
(Fig. 1A) and with different intervals of 5-aza-CdR treatment at
1
M, respectively (Fig. 1B). The cell viability was determined
by MTT assay. A dose- and duration-dependent inhibition of
cell proliferation was observed only in A549 cells but not in
H1299 and H719 cells (Fig. 1, Aand B). As shown in Fig. 1A, for
example, cell viability was decreased to 77% of the untreated
control when A549 cells were treated with 5-aza-CdR even at
very low dose (0.078
M) and 51% or 39% of the untreated
control when cells were treated with 5-aza-CdR at 1.25 or 5
M,
respectively. However, 5-aza-CdR was unable to induce an obvi-
ous decrease in cell viability in H1299 cells or H719 cells until the
concentrations were well above 1
M(Fig. 1, Aand B). Cell
viability was decreased in H1299 and H719 cells only when they
were treated at very high concentrations (10–20
M).
Inhibition of cell proliferation is a reflection of cell cycle
arrest that is mainly controlled by proteins from the INK4
family and the CIP/KIP family of cyclin-dependent kinase in-
hibitors (35). A549 cells have been reported to have a homozy-
gous deletion of CDKN2a (36). Therefore, we hypothesized that
p21
Waf1/Cip1
expression may be a critical factor for the 5-aza-
CdR-induced inhibition of cell proliferation in A549 cells. Fig.
1Cindicates that increased p21
Waf1/Cip1
expression is a dose-
dependent effect of 5-aza-CdR treatment in A549 cells but not
in H1299 cells. The increased expression of p21
Waf1/Cip1
was
observed in the A549 cells even when treated at a very low
concentration (0.01
M) of 5-aza-CdR. However, 5-aza-CdR
even at a very high concentration (10
M) was unable to induce
increased expression of p21
Waf1/Cip1
in H1299 cells (Fig. 1C).
Because p53 is a mutant in H719 cells (30), increased
p21
Waf1/Cip1
expression upon 5-aza-CdR treatment was simi-
larly not observed in H719 cells in this study (data not shown).
5-Aza-CdR-induced Inhibition of Cell Proliferation Is De-
pendent on p53/p21
Waf1/Cip1
Pathway—To investigate whether
5-aza-CdR-induced p21
Waf1/Cip1
expression is through activa-
tion of p53, a wild-type HPV E6 gene was inserted into pCMV-
neo. After transfection and selection with G418, the pCMV-
neo-E6 stable clone (A549-E6) was established. For testing the
functionality of the stable clone, the A549-E6 cells were irra-
diated with
␥
-rays, and then p53 and p21
Waf1/Cip1
levels were
determined by Western immunoblot analysis. As shown in Fig.
2A, the p53 level in A549-E6 cells was much lower than that in
A549 cells after exposure to 2 or 8 grays of
␥
-rays. Similarly,
p21
Waf1/Cip1
levels in the A549 and the A549-E6 cells after
irradiation showed the same trend as the p53 expression (Fig.
2B). These data suggested that the expression of HPV E6 in the
A549-E6 clone is sufficient to degrade the p53 level in the
transfected A549 cells. Next, A549 and A549-E6 cells were
treated with 5-aza-CdR at different concentrations for 72 h,
and then MTT assay was performed to test cell viability in a
fashion analogous to the experiments demonstrated in Fig. 1A.
As shown in Fig. 2C, cell viability in the A549-E6 was much
higher than that in the A549 cells after treatment with 5-aza-
CdR at different concentrations. These results support the
hypothesis that cell viability after treatment with 5-aza-CdR is
related to wild-type p53 in the A549 cells. To investigate the
reasons for the difference between A549 and A549-E6 cells, p53
and p21
Waf1/Cip1
expression was evaluated by Western im-
munoblot (Fig. 2, D–E). Clearly, both p53 levels as well as
p21
Waf1/Cip1
levels were significantly increased in A549 cells
after treatment with 5-aza-CdR (Fig. 2, D–E). This was not
seen in the A549-E6 cells, even when they were treated with
5-aza-CdR at higher concentrations (up to 10
M; Fig. 2E).
Although p21
Waf1/Cip1
expression is slightly increased in the
A549-E6 cells upon treatment with 5-aza-CdR, the effect is
very attenuated when compared with the parental A549 cells
(Fig. 2E).
For further confirmation of the critical role of p53 in the
5-aza-CdR-induced p21
Waf1/Cip1
expression, a full-length p21
promoter construct (pWWP-Luc) that included two p53 binding
elements and a truncated p21 promoter (pWP101-Luc) that
lacked any p53 binding site (Fig. 3A) were transiently trans-
fected into A549 cells, and relative luciferase activity (RLU)
was evaluated after treatment with 5-aza-CdR in both trans-
fectants. As shown in Fig. 3B, the RLU in the pWWP-Luc-
transfected A549 cells was much higher than that in the
pWP101-Luc-transfected A549 cells after treatment with
5-aza-CdR. The 5-aza-CdR-induced increase in the RLU was
dose-dependent. The RLU in pWWP-transfected cells, for ex-
ample, increased 3-fold when treated with 5-aza-CdR at 5
M
compared with the same transfectants treated at 0.5
M(Fig.
FIG.2. Changes in cell viability and p21
Waf1/Cip1
and p53 pro-
tein expression in A549 cells or A549-E6cells treated with 5-aza-
CdR, respectively. A549 cells or A549-E6 cells were irradiated with
␥
-rays at 2 or 8 grays, and then Western blot was performed to detect
the changes of p53 (A) and p21
Waf1/Cip
(B). A549 and A549-E6 cells were
also treated with 5-aza-CdR at different concentrations, and then MTT
assay was performed to test cell viability (C), and Western blot was
performed to detect the changes of p53 (D) and p21
Waf1/Cip1
(E).
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3B). The RLU, however, was not significantly increased in the
pWP101-transfected A549 cells after 5-aza-CdR treatments
(Fig. 3B). Because there is no endogenous p53 in H1299 cells,
the RLU was not increased in the pWWP-transfected H1299
cells after treatment with 5-aza-CdR (Fig. 3C).
To determine which p53 binding site of the p21 promoter was
important for the 5-aza-CdR treatment, pWWP-Luc containing
the mutated first p53 binding site (Mut-1) and second p53
binding site (Mut-2) were constructed (Fig. 3A) and then trans-
fected into A549 cells that were subsequently treated with
5-aza-CdR. As shown in Fig. 3D, the RLUs decreased from 3.4
to 1.42 and 1.51, respectively, after 5-aza-CdR treatment when
the Mut-1 or Mut-2 transfectants were compared with wild-
type pWWP-transfected cells (Fig. 3D). No difference in the
RLU between the Mut-1 and Mut-2 transfected cells was ob-
served after 5-aza-CdR treatment (Fig. 3D). The data suggest
that both p53 binding sites in the p21 promoter are important
in inducing p21
Waf1/Cip1
expression in response to 5-aza-CdR
treatment.
5-Aza-CdR Activates the p53/p21 Pathway through DNA
Damage—Because 5-aza-CdR is a DNA methyltransferase in-
hibitor, it was necessary to rule out the possibility that the p21
promoter is fully or partially methylated in A549 cells. To
detect the methylation status of the p21 promoter, we per-
formed combined bisulfite restriction analysis and bisulfite
sequencing of the p21 promoter from A549 and H1299 cells.
The combined bisulfite restriction analysis confirmed that the
CpG island of the p21 promoter (⫺233 to ⫹2; Fig. 3A) was
unmethylated (Fig. 4). Bisulfite sequencing analysis confirmed
these findings (Table I). Ten separate clones were selected for
the sequencing. The promoter regions of the p21 gene in A549
and H1299 cells were shown to be almost totally unmethylated
in the 24 CGs analyzed by bisulfite sequencing (1 of 240 CGs in
A549 cells and 3 of 240 CGs in H1299 cells are methylated,
respectively; Table I).
To investigate whether 5-aza-CdR plays a direct role in dam-
aging DNA, comet assay was performed as well. A549 cells
were treated with 5-aza-CdR at 0.1, 1, and 5
Mfor 72 h and
then harvested for this assay. As shown in Fig. 5, dose-depend-
ent DNA damage was observed after 5-aza-CdR treatment.
Compared with the untreated control (Fig. 5A), 5-aza-CdR even
at a low concentration (0.1
M) induced DNA damage, as indi-
cated by the presence of a DNA tail (Fig. 5B). Greater DNA tail
area and longer DNA tail length (a distance from DNA head to
the end of DNA tail) show more extensive DNA damage. These
5-aza-CdR-induced DNA-damaging features are observed more
frequently in the cells treated with a higher concentration of
5-aza-CdR than that with a lower concentration of 5-aza-CdR
(Fig. 5, compare Cand Dwith A). The quantitative data of
5-aza-CdR-induced DNA damage as determined by comet as-
say are shown in Table II. For example, the percentage of DNA
tail area relative to the total area and the DNA tail length are
increased 2.6- and 1.4-fold, respectively, when cells were
treated with 5-aza-CdR at 0.1
Mcompared with that in the
untreated cells (Table II). Similarly, in the cells treated with
5-aza-CdR at 5
M, the percentage and the length are increased
FIG.3.Changes in relative luciferase activity in A549 or H1299 cells after 5-aza-CdR treatment. A, a schematic diagram indicating p53
binding sites and CpG island of the p21 promoter. There are two p53 binding sites in the p21 promoter, GAACA (⫺2234 to ⫺2230 relative to
transcriptional start site, first binding site) and AGACT (⫺1344 to ⫺1340 relative to transcriptional start site, second binding site). The p53
binding sites were altered by site-directed mutagenesis, to GAAAC (first binding site) and AGAAT (2
nd
binding site). The region (⫺313 to ⫹552
relative to transcriptional start site) shows a range of CpG island in the p21 promoter (NCBI U24170). The dashed line (⫺233 to ⫹2 relative to
transcriptional start site) shows the area that underwent bisulfite sequencing. Band C, pWWP or truncated p21 promoter, pWP101, was
transfected into A549 (B) or H1299 cells (C). The transfected cells were then treated with 5-aza-CdR at different concentrations for 48 h. D, mutated
pWWP containing the first p53 mutated binding site (Mut-1) or the second p53 mutated binding site (Mut-2) was transfected into A549 cells and
then treated with 5-aza-CdR at 1
Mfor 48 h, respectively. The luciferase activity was normalized for the amount of protein in the cell lysate. All
of the luciferase experiments were carried out at least three times in triplicate.
Increased Expression of p21
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4.2- and 3.8-fold, respectively (Table II). These results suggest
that 5-aza-CdR inhibits cell proliferation by damaging DNA,
which causes activation of the p53/p21
Waf1/Cip1
pathway.
DISCUSSION
Increased expression of p21
Waf1/Cip1
after inhibition of DNA
methyltransferase has been reported by several investigators
(37–39). To date, at least two separate mechanisms explain this
effect. The first mechanism involves a demethylating function.
5-Aza-CdR, for example, was reported to bind to DNMT and
inactivate the enzyme (40), inducing a re-expression of
p21
Waf1/Cip1
in cells that are hypermethylated in the promoter
of the p21 gene (30, 41, 42). A second mechanism for enhanced
p21
Waf1/Cip1
expression is independent of DNA methylation.
For instance, a significant increase in p21
Waf1/Cip1
expression
was observed in human cancer T24 cells following treatment
with DNMT antisense oligonucleotides (43) or DNMT antago-
nist (37) and in normal human fibroblasts with 5-aza-CdR (44),
but the promoter region of p21 in these cells is totally unmethy-
lated. These data indicate that inhibition of DNMT itself, un-
related to methylation status, may activate p21
Waf1/Cip1
expres-
sion. Consistent with these reports, in the present study, the
5-aza-CdR-induced p21
Waf1/Cip1
expression in A549 cells is not
associated with DNA methylation because the promoter region
of p21 is almost completely unmethylated (Fig. 4 and Table I).
As one of the principle downstream effectors of p53,
p21
Waf1/Cip1
is a cyclin-dependent kinase inhibitory protein and
plays a role in preventing cyclin E/Cdk2 and cyclin A/Cdk2
kinase from promoting cell cycle progression (45). The ability of
p53 to induce cell cycle arrest in the G
1
phase in response to
DNA damage is largely dependent on p21
Waf1/Cip1
expression
(26). Therefore, the p53/p21
Waf1/Cip1
pathway confers the dam-
aged cells enough time to repair DNA and ensure that the cell
cycle progresses correctly. There is conflicting data as to
whether expression of p21
Waf1/Cip1
in cells after inhibition of
DNMT is dependent on p53 changes. 5-Aza-CdR induced an
elevated p53 level in human colon cancer cells, but p21
Waf1/Cip1
expression is not completely dependent on p53 status. In their
experimental system, Karpf et al. (27) showed that p21
Waf1/Cip1
expression induced by 5-aza-CdR was not only observed in
human colon tumor cells HCT (p53
⫹/⫹
) but also seen in HCT
(p53
⫺/⫺
) cells, although the degree of elevated p21
Waf1/Cip1
is
different in both cell lines. Milutinovic also reported that
DNMT antagonists or antisense oligonucleotides can induce
rapid expression of p21
Waf1/Cip1
in A549 cells, but p53 levels are
not changed (37). In this case, the DNMT antagonist or anti-
sense oligonucleotides are not incorporated into DNA; therefore
direct DNA damage with subsequent enhanced p53 expression
does not occur. However, in the present study, p21
Waf1/Cip1
expression in A549 cells after 5-aza-CdR treatment in limited
doses is totally dependent on p53 expression (Fig. 2, D–E). This
p53-dependent p21
Waf1/Cip1
expression is demonstrated by the
following evidence: 1) Elevated p21
Waf1/Cip1
expression after
5-aza-CdR treatment occurred only in A549 cells (with wild-
type p53) but not in H1299 cells (p53-null) (Fig. 1C); 2) 5-aza-
CdR-induced p21
Waf1/Cip1
expression in A549 cells is much de-
creased when p53 levels were diminished by transfection with
an HPV E6 gene that promotes p53 degradation (Fig. 2, D–E);
and 3) deletion and mutation analysis showed increased rela-
tive luciferase activities after 5-aza-CdR treatment in cells
with transfected full-length p21 promoter (with intact p53
binding sites) but not in those with a truncated p21 promoter
(without p53 binding sites) or full-length p21 promoter (with
mutant p53 sites) (Fig. 3, Band D). Therefore, it is reasonable
to hypothesize that the antineoplastic effect of 5-aza-CdR
TABLE I
Methylation status in A549 or H1299 cells measured
by bisulfite sequencing
Cell Methylated CG Unmethylated CG Methylation
%
A549 1 239 0.42
H1299 3 237 1.25
TABLE II
Comparison of degree of DNA damage after 5-aza-CdR treatment
using the comet assay in A549 cells
Treatment Tail area/total area DNA tail length
%
0
M17 ⫾9 18.33 ⫾2.1
0.1
M44 ⫾21 25.38 ⫾3.5
1
M58.3 ⫾1 60.07 ⫾10.2
5
M70.6 ⫾3 70.2 ⫾15
FIG.4. Methylation status analysis of the p21promoter as-
sayed by combined bisulfite restriction analysis. DNA extracted
from A549 or H1299 cells was treated with bisulfite, and then methy-
lation specific PCR was performed. Within the sequence of the PCR
product there is one TaqI site (T) and two HhaI sites (H). The PCR
product was incubated with HhaI and TaqI, respectively, for appropri-
ate times and then loaded into a polyacrylamide gel to detect methyl-
ation pattern. DNA was treated with CpG methylase and then treated
with bisulfite as a positive control. Non-bisulfite-treated DNA was used
as a negative control.
FIG.5.Detection of 5-aza-CdR DNA damage by COMET assay.
DNA damage is characterized by the percentage of DNA tail area/whole
DNA area (%) and the comet tail length (from the center of DNA head
to the end of the DNA tail) in A549 cells. The bigger the DNA tail area
(%) or the longer the DNA tail length, the more significant the damage.
A, control, the DNA is intact without DNA tail. B, 5-aza-CdR, 0.1
M.C,
5-aza-CdR, 1
M.D, 5-aza-CdR, 5
M.
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Waf1/Cip1
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may activate p53 and subsequently induce an increase in
p21
Waf1/Cip1
expression through which cell proliferation is
inhibited.
Increases in p53/p21
Waf1/Cip1
levels in mammalian cells are
often observed when cells are exposed to DNA-damaging
agents including irradiation (46, 47), UV light (48, 49), and
chemicals (50). Upon exposure to these DNA-damaging ele-
ments, p53 is activated, taking part in post-translational mod-
ifications including phosphorylation and acetylation (26, 51,
52). Previously, 5-aza-CdR was reported to be a potential DNA-
damaging agent in embryonic cells, and its cytotoxicity was
related to DNMT itself and not the secondary demethylation of
genomic DNA (15). 5-Aza-CdR was incorporated into DNA, and
then the spontaneous degradation of the incorporated analog
may result in DNA damage (11, 53). It has also been shown
that 5-aza-CdR, when incorporated into DNA, covalently links
with DNMT, an event that could cause DNA damage because of
the structural instability at its incorporation sites (54) or by
obstructing DNA synthesis (15, 55). Consistent with these re-
ports, our data for the first time demonstrates directly that
5-aza-CdR is a DNA-damaging agent when assayed by comet
assay (Fig. 5 and Table II).
Interestingly, growth inhibition and cytotoxicity induced by
5-aza-CdR at higher doses (⬎20
M) are not dependent on p53
status because the cells tested are mostly dead after 5-aza-CdR
treatment (data not shown). This p53-dependent inhibition of
cell proliferation by a limited dose of 5-aza-CdR treatment has
a great potential advantage in the treatment of different hu-
man cancers. The major reported side effect of 5-aza-CdR when
pushed to the maximum tolerated dose is hematopoietic toxic-
ity (10). A more limited dose of 5-aza-CdR treatment may
decrease these toxic effects on normal tissue and cells. In ad-
dition, the limited dose of 5-aza-CdR required to induce effects
in wild-type p53 cells may explain why different human cancer
cells appear to have different sensitivities to 5-aza-CdR treat-
ment. p53 status may be a critical factor for judging the antin-
eoplastic effects of 5-aza-CdR in cancer cells. Although there is
insufficient data to show a relationship between p53 status and
efficacy of 5-aza-CdR in patients, several studies suggest that
p53 status may be a key predictive factor for the efficacy of
5-aza-CdR treatments (10, 24, 56). For example, 5-aza-CdR
appears to be very effective in the treatment of adult human
chronic myelogenous leukemias, which generally have wild-
type p53 (56, 57) but is less demonstrably effective in the
treatment of solid tumors, such as human lung cancers, which
typically have mutant p53 (24, 58). Our data suggest that the
future clinical development of 5-aza-CdR may depend on ge-
netic factors such as the p53 status of the treated tumor.
Acknowledgments—We thank Drs. T. Sakai and H. Ding for provid-
ing us with the vectors used in this study. We also thank Dr. Y. Shang
and Quanhui Zhen, Shan Chen, and Zhe Li for technical help. We
appreciate Dr. X. B. Yao (University of Science and Technology of
China) for support and encouragement during the course of this study.
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