From the Department of Pharmacology and Therapeutics,
McGill University, Montreal, Quebec H3G 1Y6, Canada and
¶ Laboratoire de Virologie Moléculaire et Structurale, EA
2939, Faculté de Médecine et Pharmacie, Université
Joseph Fourier de Grenoble, Domaine de La Merci, Ave.
Gresivaudan, La Tronche 38706, France
Received for publication, December 27, 2002, and in revised form, February 5, 2003
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ABSTRACT |
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The DNA methylation pattern is an important
component of the epigenome that regulates and maintains gene expression
programs. In this paper, we test the hypothesis that vertebrate cells
possess mechanisms protecting them from epigenomic stress similar to
DNA damage checkpoints. We show that knockdown of DNMT1
(DNA methyltransferase 1) by an antisense oligonucleotide triggers an
intra-S-phase arrest of DNA replication that is not observed with
control oligonucleotide. The cells are arrested at different positions
throughout the S-phase of the cell cycle, suggesting that this response
is not specific to distinct classes of origins of replication. The
intra-S-phase arrest of DNA replication is proposed to protect the
genome from extensive DNA demethylation that could come about by
replication in the absence of DNMT1. This protective
mechanism is not induced by 5-aza-2'-deoxycytidine, a nucleoside
analog that inhibits DNA methylation by trapping DNMT1 in the
progressing replication fork, but does not reduce de novo
synthesis of DNMT1. Our data therefore suggest that the intra-S-phase
arrest is triggered by a reduction in DNMT1 and not by demethylation of
DNA. DNMT1 knockdown also leads to an induction of a set of genes that
are implicated in genotoxic stress response such as
NF- Proper epigenomic regulation of gene expression is essential for
the integrity of cell function. One critical component of the epigenome
is the pattern of distribution of methylated cytosines in CG
dinucleotide sequences in the genome (1). Methylation of
CGs marks genes for inactivation by either interfering with the binding
of methylated DNA-sensitive transcription factors (2) or by
recruiting methylated DNA-binding proteins such as MeCP2, which in turn
recruit corepressor complexes and histone deacetylases to the chromatin
associated with the gene (3). The methylation pattern can thus
determine the chromatin structure and state of activity of genes.
Disruption in the proper maintenance of the DNA methylation pattern
results in aberrant gene expression, as is observed in tumor suppressor
genes that are hypermethylated in cancer (4). Aberrant
hypomethylation can also result in improper activation of genes
(5).
The main enzyme responsible for replicating the DNA methylation pattern
is DNMT1 (DNA methyltransferase
1). This enzyme shows preference for hemimethylated DNA and
is therefore believed to faithfully copy the DNA methylation pattern
(6). Multiple mechanisms have been proposed to coordinate the
inheritance of DNA methylation patterns with DNA replication. First,
DNMT1 expression is regulated with the cell cycle (7, 8), and it is
up-regulated by proto-oncogenes Ras and Jun (9-11), Fos (12), and T
antigen (13). Second, DNMT1 is localized to the replication
fork (14) and is associated with the replication protein proliferating
cell nuclear antigen (15). Third, DNA methylation occurs
concurrently with DNA replication (16). This temporal and physical
association of DNMT1 with DNA replication is believed to have evolved
to guarantee concordant replication of DNA and its methylation pattern.
Previous studies have shown that inhibition of DNMT1 can lead to
inhibition of initiation of DNA replication (17), but it is not clear
whether this response is a consequence of induction of tumor suppressor genes such as p21 (18) or p16 (19), leading to retreat from the cell
cycle. A conditional knockout of murine dnmt1 gene was also
shown to reduce the rate of cell division (5), but it is still unclear
whether inhibition of DNMT1 leads to a change in cell cycle kinetics
similar to DNA damage response checkpoints.
Multiple mechanisms have been established to guard the integrity of the
genome in response to DNA damage. For example, two parallel,
cooperating mechanisms, both regulated by ATM, jointly contribute to
the rapid and transient inhibition of firing of origins of DNA
replication in response to ionizing radiation (20-22). This stalling
of DNA synthesis is required to prevent genetic instability by
coordinating replication and repair. We reasoned that similar
mechanisms guard the integrity of epigenomic information in response to
a disruption in the DNA methylation machinery.
In this paper, we test this hypothesis by determining the response of
human cell lines to a knockdown of DNMT1 mRNA, encoding the enzyme responsible for the replication of the DNA methylation pattern. Our data suggest that cells respond to this epigenomic stress
by an intra-S-phase arrest of DNA synthesis as well as by inducing a
large number of stress response genes. The slow down in DNA synthesis
during S-phase protects the DNA from a global loss of the methylation
pattern. This mechanism is not triggered by 5-aza-2'-deoxycytidine
(5-aza-CdR),1 which causes an
extensive loss of DNA methylation.
Cell Culture, Antisense Oligonucleotides, and 5-aza-CdR
Treatment--
Both A549, a human non-small cell lung carcinoma cell
line, and T24, a human bladder transitional carcinoma-derived cell
line, were obtained from the ATCC (Manassas, VA). A549 cells were grown in Dulbecco's modified Eagle's medium (low glucose) supplemented with
10% fetal calf serum and 2 mM glutamine. T24 cells were
maintained in McCoy's medium supplemented with 10% fetal calf serum,
2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml
streptomycin. 18-24 h prior to treatment, cells were plated at a
concentration of 3 × 105 cells/100-mm tissue culture
dish or 5 × 104 cells/well in a six-well plate in the
absence of antibiotics. The phosphorothioate oligodeoxynucleotides
used in this study were MG88 (human DNMT1 antisense
oligonucleotide) and its mismatch control MG208, which has a 6-base
pair difference from MG88 (19). Oligonucleotides were transfected into
cells with 6.25 µg/ml Lipofectin (Invitrogen) in serum-free Opti-MEM
(Invitrogen). The oligonucleotide-containing Opti-MEM medium was
removed from the cells and replaced with regular growth medium after
4 h. The treatment was repeated every 24 h. The cells were
harvested 24, 48, 72, and 96 h following the first transfection.
For 5-aza-CdR treatment, cells were grown in regular culture medium in
the presence of 10 DNA Methyltransferase Activity Assay and Western Blot
Analysis--
To determine the level of cellular DNA methyltransferase
activity, nuclear extracts were prepared, and DNA methyltransferase activity was assayed as described previously (8). For Western blot
analysis of DNMT1, 50 µg of nuclear protein was fractionated on a 5%
SDS-polyacrylamide gel, transferred to polyvinylidene difluoride
membrane, and reacted with the polyclonal anti-DNMT1 antibody (New
England Biolabs) at a dilution of 1:2000 in the presence of 0.05%
Tween and 5% milk, and it was then reacted with anti-rabbit IgG
(Sigma) at a dilution of 1:5000. The amount of total protein per lane
was determined by Amido Black staining (23). The intensity of DNMT1 and
total protein signal was measured by scanning densitometry, and the
ratio of DNMT1/total nuclear protein was calculated.
RT-PCR--
Total RNA was extracted using the standard guanidium
isothiocyanate method (24). cDNA was synthesized in a 20-µl
reaction volume containing 2 µg of total RNA, 40 units of Moloney
murine leukemia virus reverse transcriptase (MBI), 5 µM
random primer (Roche Molecular Biochemicals), a 1 mM
concentration of each of the four deoxynucleotide triphosphates, and 40 units of RNase inhibitor (Roche Molecular Biochemicals). mRNA was
denatured for 5 min at 70 °C, the random primers were annealed for
10 min at 25 °C, and mRNA was reverse transcribed for 1 h
at 37 °C. The reverse transcriptase was heat-inactivated for 10 min
at 70 °C, and the products were stored at
PCR was performed in a 50-µl reaction mixture containing 3 µl of
synthesized cDNA product, 5 µl of 10× PCR buffer, 1.5-2.0 mM MgCl2, 0.2 mM dNTP, 1 unit of
Taq polymerase (all from MBI) and 0.5 µM of
each primer. The primer sequences that were used for the different
mRNAs were GADD45 Competitive PCR for Quantification of DNMT1 mRNA
Levels--
Total RNA (2 µg) was reverse transcribed as described
above in the presence of 12.5 µCi of 35S-labeled dCTP
(1250 Ci/mmol) (ICN) to quantify the efficiency of reverse
transcription. Equal amounts of reverse transcribed cDNA (70,000 cpm as determined by the incorporation of 35S-labeled dCTP)
were subjected to PCR amplification in the presence of increasing
concentrations of a competitor DNA fragment that amplifies with the
same set of primers but yields a product that is shorter by 48 base
pairs. The following primers were used: 5'-ACCGCTTCTACTTCCTCGAGGCCTA-3'
(DNMT1 sense),
5'-GTTGCAGTCCTCTGTGAACACTGTGG-3'(DNMT1 antisense), and
5'-CGTCGAGGCCTAGAAACAAAGGGAAGGGCAAG (primer used to generate the
competitor). PCR conditions were as follows: 94 °C for 1 min,
65 °C for 1 min, and 72 °C for 1 min (33 cycles).
Methylation-specific PCR--
Genomic DNA was extracted with DNA
extraction buffer (1% SDS, 5 mM EDTA, 150 mM
NaCl) followed by proteinase K digestion, phenol/chloroform extractions
and ethanol precipitations. Bisulfate treatment was performed as
described previously (25). The methylation status of the p16
gene was determined by methylation-specific PCR (26) as modified by
Palmisano et al. (27).
[3H]Thymidine Incorporation Assay--
Cells were
plated in a six-well plate (5 × 104/well). For the
final 4 h of incubation, 1 µCi/ml
[methyl-3-3H]thymidine (PerkinElmer Life
Sciences) was added to the medium. After washing twice with PBS, the
cells were incubated in 10% trichloroacetic acid for 30 min at
4 °C, washed twice with cold 10% trichloroacetic acid, and then
lysed with 1 N NaOH and 1% SDS.
[3H]thymidine incorporation was measured using a liquid
scintillation counter (LKB Wallac).
Flow Cytometry Analysis of 5-Methylcytosine Staining--
Global
DNA methylation was evaluated by staining the cells with specific
monoclonal antibody against 5-methylcytidine using the protocol
described previously (28) with slight modifications. Briefly, cells
were washed with phosphate-buffered saline (PBS) supplemented with
0.1% Tween 20 and 1% bovine serum albumin (PBST-BSA), fixed with
0.25% paraformaldehyde at 37 °C for 10 min and 88% methanol at
Microarray Analysis--
A549 cells were transfected with 200 nM MG208 or MG88 or were treated with 1 µM
5-aza-CdR or Me2SO, for 48 h. Total RNA was extracted
with RNAeasy (Qiagen). Micoarray analysis was performed as previously
described (29). Briefly, 20 µg of RNA was used for cDNA
synthesis, followed by in vitro transcription with a T7
promoter primer having a poly(T) tail. The resulting product was
hybridized and processed with the GeneChip system (Affymetrix) to a
HuGeneFL DNA microarray containing oligonucleotides specific for
~12,000 human transcripts. Data analysis, average difference, and
expression for each feature on the chip were computed using Affymetrix
GeneChip Analysis Suite version 3.3 with default parameters. The gene
expression analysis was performed by the Montreal Genome Center.
Double Staining of BrdUrd and Propidium Iodide--
Cells were
incubated with 10 µM BrdUrd (Sigma) for the last 2 h
before harvesting. Incorporated BrdUrd was stained with anti-BrdUrd antibody conjugated with fluorescein isothiocyanate (Roche Molecular Biochemicals) following the manufacturer's protocol (30). After the
last washing, the cells were resuspended in PBS containing 50 µg/ml
propidium iodide and 10 µg/ml RNase A for 30 min at room temperature
and then analyzed with FACScan (BD Bioscience) for both fluorescein
isothiocyanate and propidium iodide fluorescence.
Knockdown of DNMT1 mRNA by the DNMT1 Antisense Oligonucleotide
MG88--
DNMT1 activity is physically and temporally associated with
the DNA replication machinery. The absence of DNMT1 from the
replication fork could potentially lead to an epigenomic catastrophe.
We have previously proposed that the coordination of DNMT1 expression and DNA replication evolved as a mechanism to protect the coordinate inheritance of genetic and epigenetic information (31, 32). To test
this hypothesis, we determined the cellular response to a knockdown of
DNMT1 protein. We took advantage of a previously described antisense
oligonucleotide, which specifically knocks down DNMT1
mRNA (19) MG88 and its mismatch control MG208 (see Fig.
1A for sequence and alignment
with human and mouse DNMT1 mRNA). We first optimized the time and
concentration at which MG88 specifically knocks down DNMT1
activity in A549 cells in comparison with MG208. The results presented
in Fig. 1B show that MG88 reduces DNA methyltransferase
activity in a dose- and time-dependent manner relative to
MG208. Inhibition of DNA methyltransferase activity approximates
80% after 48 h of MG88 treatment, whereas no inhibition is
observed following MG208 treatment. Therefore, for our further analysis
we chose to treat the cells with a 200 nM concentration of
either MG88 or MG208 for 48 h. We confirmed the antisense
mechanism of action of MG88 by demonstrating that DNMT1
mRNA levels are knocked down following a 48-h treatment with this
oligonucleotide in comparison with MG208 treatment using a competitive
RT-PCR assay for DNMT1 (Fig. 1, C and
D). To confirm that DNMT1 inhibition results in reduction of
DNMT1 protein, nuclear extracts prepared from either MG208- or
MG88-treated cells (200 nM for 48 h) were subjected to
a Western blot analysis and reacted with anti-DNMT1 antibody (Fig.
1E). Quantification of the signal by densitometry reveals
85% reduction in protein levels. Inhibition of DNMT1
mRNA by MG88 was also confirmed in a gene array expression analysis
presented in Table II.
DNMT1 Knockdown Reduces the Fraction of Cells That Are in
S-phase--
Several studies have previously demonstrated that
inhibition of DNMT1 results in inhibition of cell growth (33). One
possible explanation for the reduced cell growth is that knockdown of
DNMT1 results in inhibition of firing of DNA replication origins (17). We therefore addressed the question of whether this inhibition of DNA
replication reflects a distinct alteration in cell cycle kinetics,
similar to the DNA damage checkpoints that trigger arrest at distinct
phases of the cell cycle (34).
A549 cells were treated with a 200 nM concentration of
either MG88 or MG208 for 24-96 h as described under "Materials and Methods." As observed in Fig. 2,
DNMT1 knockdown results in a significant decrease in overall
DNA synthetic capacity of A549 cells. This is illustrated by the
reduced incorporation of [3H]thymidine into DNA 24 h
after the initiation of treatment, as has been previously reported
(17). To exclude the possibility that inhibition of DNA synthesis by
MG88 is independent of DNMT1 expression and is a toxic side effect of
the sequence, we took advantage of the species specificity of MG88. As
shown in Fig. 1A, there is a 6-base pair mismatch between
MG88 and the mouse dnmt1 mRNA. We therefore determined
whether MG88 would inhibit DNA synthesis in a mouse adrenal carcinoma
cell line, Y1, which was previously shown by us to be responsive
to a mouse dnmt1 antisense oligonucleotide (23). The results
shown in Fig. 2B demonstrate that 48-h treatment with 200 nM MG88 had no significant impact on the DNA synthetic
capacity of Y1 cells in comparison with MG208, supporting the
hypothesis that MG88 inhibition of DNA synthesis is
DNMT1-dependent.
We then addressed the question of whether this inhibition in DNA
synthesis represents a slowdown in the rate of DNA synthesis or a
reduction in the fraction of cells that are in the synthetic phase,
which would indicate a change in cell cycle phase kinetics. We pulsed
MG88- and MG208-treated cells with BrdUrd 48 h after the
initiation of treatment and sorted the cells that incorporated BrdUrd
using fluorescence-activated cell sorting as described under
"Materials and Methods." As illustrated in Fig. 2C,
DNMT1 knockdown reduces the fraction of cells that incorporate DNA (the M1 population). However, the reduction in the fraction of cells that
synthesize DNA (up to 42%) does not account for the overall reduction
in DNA replication shown by the [3H]thymidine
incorporation assay, which is >95%. Although there is no significant
cell death that can account for this disparity, this difference might
reflect the fact that cell number following MG88 treatment is reduced.
DNMT1 Knockdown Results in Intra-S-phase Arrest in DNA
Replication--
The reduction in the fraction of cells that
incorporate DNA could be a consequence of a phase specific cell cycle
arrest. However, preliminary results using flow cytometry sorting of
propidium iodide-stained cells failed to show either a significant
G1 or G2 arrest that could explain the
reduction in S-phase cells. This raised the possibility that
DNMT1 knockdown caused an intra-S-phase arrest similar to
the previously described DNA damage checkpoint (35). To address this
possibility, we treated A549 cells for 48 h with either MG88 or
MG208 and then pulsed with BrdUrd to mark cells that are actively
replicating. We then stained the cells with propidium iodide to
determine their total DNA content, which is indicative of their
position in the cell cycle. The cells were analyzed simultaneously for
both their BrdUrd incorporation and propidium iodide staining by flow
cytometry. The results of a representative analysis shown in Fig.
3A suggest a slight increase in sub-G1 population and a slight increase in cells found
in the G2 phase of the cell cycle in response to
DNMT1 knockdown (9.76% in MG88-treated cells
versus 7.71% in the MG208 treatment). However, an
unexpected change in the cell cycle kinetics is a consequence of an
intra-S-phase DNA replication arrest. This arrest results in the
partition of cells found in the S phase into two distinct populations, one that incorporates BrdUrd and another comprising 30%
of the cells in the S-phase that do not incorporate BrdUrd, in
comparison with less than 7% of those in the MG 208 control. Similar
observations were obtained in five independent experiments. The
presence of a population of cells in S-phase that does not incorporate
BrdUrd in response to depletion of DNMT1 is consistent with a presence
of an intra-S-phase checkpoint triggering cell cycle arrest. Cells that
do not incorporate BrdUrd in S-phase are distributed throughout the
S-phase of the cell cycle, indicating that the arrest in DNA
replication does not occur at a specific point in S-phase. DNMT1
knockdown can lead to intra-S-phase arrest at any point in S-phase. As
has been previously observed for DNA damage checkpoints, the
intra-S-phase arrest is transient, since this partition of the S-phase
cell population disappears after longer treatment (10 days) (Fig.
3B).
The Intra-S-phase Arrest Is Not Dependent on the Extent of DNA
Demethylation--
The intra-S-phase arrest in DNA replication might
have been triggered by either the absence of DNMT1 from some
replication forks or by demethylated DNA. To address this question, we
took advantage of a well characterized inhibitor of DNA methylation, 5-aza-CdR (36). 5-aza-CdR is a nucleoside analogue that is incorporated into DNA following its phosphorylation to the trinucleotide form. It
inhibits DNA methylation only once it is incorporated into DNA by
trapping DNMT1 from the progressing replication fork. 5-aza-CdR does
not inhibit either de novo synthesis of DNMT1 or its
incorporation into the replication fork, but it inhibits DNA
methylation during replication. It is dependent on DNA replication for
its action, in contrast to DNMT1 antisense, which reduces
the availability of DNMT1 prior to the formation of the replication fork.
5-aza-CdR causes extensive DNA demethylation in A549 cells as well as
many other cell lines and should cause a drastic change in cell cycle
kinetics in S-phase if the trigger for intra-S-phase arrest in DNA
replication is DNA demethylation. However, whereas 5-aza-CdR treatment,
using a concentration that causes significant demethylation, results in
a limited decrease in the rate of DNA synthesis as indicated by
thymidine incorporation analyses shown in Fig.
4A, this inhibition is
considerably less than that observed with MG88 (Fig. 2A),
and significant DNA synthesis occurs up to 96 h after treatment
with 5-aza-CdR. Similarly, the analysis of the distribution of
BrdUrd-incorporating cells in S (Fig. 4B) does not show
a clear partition into two distinct populations as has been observed
with MG88. 5-aza-CdR-treated cells show a gradual limited decrease in
DNA replication rate as indicated by the gradient of BrdUrd-labeled
cells in the S phase of the cell cycle. The continued synthesis of DNA
in the presence of 5-aza-CdR can explain the ability of this agent to
demethylate DNA. The main change in the cell cycle kinetics that is
observed with 5-aza-CdR is an increase in the G2 population
(from 9.74% in the control to 28.99% in 5-aza-CdR-treated cells).
These results suggest that the intra-S-phase arrest is not correlated
to the degree of inhibition of DNA methylation per se. Our
data are consistent with the hypothesis that the intra-S-phase arrest
following DNMT1 knockdown is a response to a reduction in
the availability of DNMT1 in the replication fork rather than DNA
demethylation.
Intra-S-phase Arrest of DNA Replication Possibly Protects the
Genome from Global Hypomethylation--
One potential role of the
intra-S-phase arrest triggered by reduction of DNMT1 is to protect the
epigenome from global loss of the DNA methylation pattern. Using
anti-5-methylcytosine antibodies that were previously described (37),
we compared the state of methylation of A549 cells treated for 48 h with either 200 nM MG88, which causes intra-S-phase
arrest or 5-aza-CdR which does not trigger a distinct intra-S-phase
arrest. A549 cells treated with either the DNMT1 inhibitors (MG88 or
5-aza-CdR) or their respective controls (MG208 or Me2SO)
were stained with either the 5-methylcytosine antibody or the secondary
antibody alone as a control and were subjected to
fluorescence-activated cell sorting analysis (Fig.
5, A for MG88
versus MG208 and B for 5-aza-CdR versus Me2SO control). MG88-treated cells are
only slightly demethylated, as indicated by the slight shift in the
fluorescence intensity of the MG88-treated cells (Fig. 5A),
whereas 5-aza-CdR treatment results in extensive reduction in staining
with the anti-5-methylcytosine antibody indicative of genome-wide
demethylation (Fig. 5B). The intra-S-phase arrest of
replication following MG88 treatment possibly protects A549 cells from
genome-wide loss of methylation. We have previously observed that
hemimethylated inhibitors of DNMT1 that inhibit DNA replication also
cause only limited demethylation of DNA (33).
Comparison of the Kinetics of Demethylation of the Tumor Suppressor
p16 following DNMT1 Knockdown by MG88 and 5-aza-CdR Trapping of
DNMT1--
We addressed the question of whether this difference in the
kinetics of global DNA demethylation between 5-aza-CdR and
DNMT1 antisense oligonucleotides is also observed when
specific genes are examined. We focused on the methylated tumor
suppressor gene p16 in the human bladder carcinoma cell line T24, since
there is no well documented example of a methylated gene in A549 cells that is activated by pharmacological demethylation. The p16 gene is
demethylated in response to both 5-aza-CdR (38) and DNMT1 antisense (MG88) treatment (19).
We first verified that the DNMT1 antisense-triggered
intra-S-phase arrest demonstrated above in A549 cells (Fig. 3) is also functional in T24 cells. A 48-h treatment of T24 cells with MG88 results in an intra-S-phase arrest of DNA replication (Fig.
6A) similar to that observed
in A549 cells (Fig. 3), as indicated by the partition of cells in
S-phase of the cell cycle to two distinct groups, those that
incorporate BrdUrd (9.77% compared with 37.06% in the MG208 control
group) and those that do not incorporate BrdUrd (12.4 versus
5.5% in the control). Thus, 56% of the cells found in the S-phase of
the cell cycle do not synthesize DNA following MG88 treatment of T24
cells. On the other hand, 5-aza-CdR treatment results in an increase in
the fraction of cells that are in G1 (72.06% in
5-aza-CdR-treated versus 53.55% in the control) and a
slowdown of the rate of DNA synthesis in S-phase cells, as indicated by
the gradient of the intensity of BrdUrd incorporation. However, there
is no distinct partition of the population of S-phase cells following
5-aza-CdR to two distinct groups as is observed following MG88
treatment.
We then assessed the global state of methylation of T24 cells following
either MG88 or 5-aza-CdR treatment for 48 h using fluorescence-activated cell sorting analysis of 5-methylcytosine antibody-stained cells. Fig. 6B demonstrates that similar to
what is observed in A549 cells, MG88 treatment results in very limited global hypomethylation in T24 cells, as indicated by the slight shift
to the left in the intensity of staining with 5-methylcytosine antibodies. In contrast, 5-aza-CdR results in global hypomethylation as
indicated by the considerable shift to the left of the population of
5-aza-CdR-treated cells Fig. 6C.
We then determined the pattern of methylation of the p16 gene following
either DNMT1 antisense or 5-aza-CdR treatments. The methylation pattern of the 5' exon of p16 was studied by
methylation-specific PCR that was previously described (26) (Fig. 6,
D and E). The results of this analysis show a
dramatic difference in the kinetics of demethylation between
MG88-treated (Fig. 6D) and 5-aza-CdR-treated (Fig.
6E) cells. Whereas p16 is significantly demethylated 24 h after the initiation of 5-aza-CdR treatment and is completely demethylated after 96 h (Fig. 6E), p16 remains fully
methylated 48 h after MG88 treatment at the peak of the
intra-S-phase DNA replication arrest (Fig. 6D).
Demethylation initiates only at 72 h. The mechanism of this
demethylation is unclear, since passive demethylation requires DNA
replication in the absence of DNA methyltransferase, whereas DNA
replication is inhibited in T24 cells following MG88 treatment. It is
possible that the demethylation of p16 is caused by an active
demethylation mechanism or it might result from residual replication in
the absence of DNMT1.
In summary, our data reveal that demethylation is delayed when DNA
synthesis is arrested concomitantly with knockdown of DNMT1. The signal for the intra-S-phase arrest following DNMT1
knockdown by MG88 is neither the extent of DNA demethylation nor the
activation of p16, since DNA replication arrest precedes demethylation.
Furthermore, 5-aza-CdR, a potent inhibitor of DNA methylation that acts
by a different mechanism than MG88, does not cause an intra-S-phase arrest in DNA replication.
Knockdown of DNMT1 by Antisense, but Not DNA Methylation Inhibition
with 5-aza-CdR, Induces Expression of Genotoxic Stress-responsive
Genes--
Multiple genes have been shown in the past to be silenced
by DNA methylation. A well accepted model is that global DNA
demethylation results in misprogramming of gene expression by aberrant
activation of genes that are normally silenced by methylation. A
methodical analysis of genes that are induced following 5-aza-CdR
treatment of a colorectal cancer cell line identified a group of genes
that are silenced by DNA methylation and are demethylated by 5-aza-CdR. In addition, another group of genes that are not methylated and are
activated by methylation-independent mechanisms were also shown to be
induced by 5-aza-CdR (39). We used Affymetrix 12K gene microarrays to
repeat this analysis in our system. We compared the gene expression
profile of A549 cells treated with 1 µM 5-aza-CdR with
the gene expression profile of A549 cells treated with
Me2SO for 48 h. The list of genes induced more than
2.5-fold by 5-aza-CdR is shown in Table
I. Only genes that were induced in two
separate experiments and did not show variation in expression within
either the control or 5-aza-CdR replicates were included. The list of genes induced by 5-aza-CdR includes tissue-specific genes such as
smooth muscle actin
The up-regulation of genes identified by gene array analysis was
verified by semiquantitative RT-PCR shown in Fig.
7. The cancer/testis-specific genes show
a typical profile for methylated genes induced by demethylating agents.
They are completely silenced in the control cells and are activated to
clearly detectable levels following demethylation (Fig. 7). Methylation
results in most cases in silencing of genes rather than a quantitative
reduction in gene expression. In addition, expression levels are
increased with time, as expected from passive demethylation kinetics.
Inhibition of DNA methyltransferase during new DNA synthesis results in
a time-dependent increase in the relative abundance of the
population of newly replicated unmethylated DNA. On the other hand,
genes such as BIK, which is expressed in control cells, are transiently induced by 5-aza-CdR, and their level of induction is reduced with time
(Fig. 7). This profile of induction is consistent with a
methylation-independent mechanism, which is also supported by the
induction of BIK after DNMT1 knockdown before any significant global
demethylation is observed (2-fold induction 24 h after antisense
treatment). BIK and other genes induced by a methylation-independent mechanism were also induced by the deacetylase inhibitors trichostatin A (39) and n-butyrate (41). In accordance with previous
studies in colorectal cancer (39), our data show both
methylation-dependent and -independent induction by
5-aza-CdR in A549 cells (MAGEB2, SSX2, and BIK, respectively).
Based on the data presented above, we predicted that in contrast to the
response to 5-aza-CdR, knockdown of DNMT1 by antisense inhibition
should not result in induction of methylation-silenced genes at the
early time after treatment. However, since we have previously shown
that inhibition of DNMT1 induces the expression of the p21 tumor
suppressor gene by a mechanism that does not involve DNA demethylation
(18), and since examples of such genes were identified in the recent
analysis with 5-aza-CdR (39), we tested the possibility that the early
response to DNMT1 knockdown results in a programmed change in gene
expression that precedes global hypomethylation and is possibly
involved in the stress response. We therefore compared the gene
expression profile of MG88-treated A549 cells with the gene expression
profile of A549 cells treated with the control MG208 oligonucleotide
for 48 h using Affymetrix 12K gene microarrays.
We compared the normalized gene expression profile of the two treatment
groups. 255 (2.1%) genes out of 12,000 genes were up-regulated,
whereas there were just 23 (0.19%) genes that were down-regulated. The
experiment was repeated with similar results. DNMT1
expression was 75% down-regulated in two experiments, which is an
internal validation of our gene expression analysis and antisense
treatment. The results are presented in Table
II. Only genes induced in both
experiments are included. Among the genes that were induced, we did not
identify genes that were previously characterized to be silenced by
methylation such as SSX2 and MAGEB2. These two
genes were shown to be induced with 5-aza-CdR but were not induced
following DNMT1 knockdown (Figs. 7 and
8). However, a distinct group of genes
that stood out was a set of previously characterized
genotoxic-responsive genes such as ATF-3,
GADD45 Multiple mechanisms regulate expression of DNMT1 within a cell (7,
8, 11, 32). In this paper, we address the question of whether mammalian
cells possess a mechanism to respond to a sudden loss of DNMT1 and
protect themselves from a global loss of DNA methylation during
replication in the absence of DNMT1. It is well established that
genotoxic challenges such as DNA damage evoke distinct cellular
responses, resulting in a transient intra-S-phase arrest in DNA
replication (20-22). This intra-S-phase arrest guards against buildup
of mutations during DNA replication before the other checkpoints at
G2/M and G1/S could take effect. Similarly, relying on G2/M and G1/S checkpoints to respond
to the absence of DNMT1 in the fork during replication could result in
a significant loss of DNA methylation and a buildup of epigenomic errors.
We demonstrate here that following DNMT1 knockdown, cells found in the
S phase of the cell cycle are partitioned into two groups, those that
incorporate BrdUrd and those that do not incorporate BrdUrd (Fig. 3).
Our data is consistent with the presence of an intra-S-phase checkpoint
that arrests all of the replication forks in a cell, as illustrated by
the appearance of a group of cells in S-phase that do not
incorporate any BrdUrd as a response to a reduction in availability of DNMT1.
Different origins of replication replicate at discreet and well defined
positions in the cell cycle. Origins that replicate early in the cell
cycle are associated with genomic regions that are hypomethylated and
are actively transcribed (52), whereas origins that replicate late in
S-phase are associated with inactive genes, which are also known to be
hypermethylated (53). The results presented in Figs. 3 and 6 show that
DNA replication is arrested at any point in the S-phase of the cell
cycle. This is inconsistent with the hypothesis that DNMT1 knockdown
affects only specific classes of origins.
What is the signal that triggers a stress response to antisense
DNMT1 knockdown? It is possible that the emergence of
demethylated DNA caused by replication in the absence of DNMT1 triggers
the intra-S-phase arrest. Alternatively, the signal is the inhibition of de novo synthesis of DNMT1, leading to its absence from
DNA replication factories. The fact that 5-aza-CdR, which causes a far
more extensive demethylation than MG88, does not trigger the same
magnitude of intra-S-phase arrest of replication (Fig. 3), suggests
that it is not the demethylation that triggers the intra-S-phase arrest. Rather, our data are consistent with the hypothesis that it is
the reduction in DNMT1 protein that triggers the intra-S-phase arrest
observed after MG88 treatment.
We propose that the intra-S-phase arrest guarantees that no DNA is
synthesized in the absence of DNMT1. However, this is a transient and
incomplete protection, and delayed demethylation is observed
following extended MG88 treatment (Fig. 6). 5-aza-CdR bypasses this
checkpoint to a large extent, since it does not reduce DNMT1 synthesis
but traps DNMT1 only once the replication fork has formed in the
presence of DNMT1.
These differences in the mechanisms of action of these two inhibitors
have important implications on the design and therapeutic utility of
different DNA methylation inhibitors (31). Agents such as MG88, that
reduce the availability of DNMT1 at the replication fork, are strong
inhibitors of cell growth and should be effective in inhibiting tumor
growth but will not cause extensive demethylation (33). There might be
an advantage for therapeutic agents that do not cause extensive
demethylation, since extensive hypomethylation has been previously
associated with metastasis (54) and possibly induction of silenced
repetitive elements (55, 56). The data presented in Table I illustrate
the risks inherent in using DNA-demethylating agents. In addition to
induction of antimitotic and proapoptotic genes, 5-aza-CdR induces
three families of testis/cancer-specific antigens that were previously
implicated in tumor progression and potentially tumor invasion and
metastasis. It is interesting to note that expression of the GAGE
antigen family has been associated with poor prognosis in some cancers.
G antigen 7 is expressed in prostate cancer (43), and G antigen 7c was
proposed to be an antiapoptotic gene (57). Similarly, MAGE expression
is associated with metastasis (53, 54). SSX2 was shown to be expressed
in a wide variety of tumors (50) and was identified as one of 13 antigens that react exclusively with sera from colon cancer patients but not with sera from normal patients (58).
In addition to the change in cell kinetics, the cells respond to DNMT1
knockdown by a change in the gene expression program. A significant
fraction of the induced genes is known to be involved in genotoxic
stress responses (Table II). We have previously proposed that DNMT1
controls the expression of certain genes by a direct repression
function that does not involve DNA methylation (18). DNMT1 was
previously shown to interact with HDAC1 (59), HDAC2 (60), and Rb-E2F1
(61). We propose that some of the early genes induced by DNMT1
knockdown are similarly controlled by the DNA methylation-independent
gene repression activities of DNMT1.
It remains to be seen whether the genes induced by DNMT1 knockdown are
also involved in the intra-S-phase arrest or whether they are parallel
responses that augment the protection against epigenomic loss. Recent
studies have identified some of the players involved in the
intra-S-phase checkpoint in response to ionizing DNA damage. ATM is
activated by ionizing radiation, which in turn activates two signaling
pathways, one leading to inactivation of Cdk2 and intra-S-phase
arrest and the other leading to activation of p21 and G1
arrest (35). We have previously shown that DNMT1 inhibition can lead to
transcriptional induction of p21, and here we show induction of
GADD45 Although the precise mechanism by which reduction of DNMT1 causes
intra-S-phase arrest is unknown, our data describe a new class of
putative checkpoints that react to epigenomic stress caused by
reduction of DNMT1 levels. We propose that this mechanism has evolved
to protect the genome from unscheduled demethylation and to maintain
the coordination of replication of the genome and the epigenome.
B, JunB, ATF-3,
and GADD45
(growth arrest
DNA damage 45
gene). Based on
these data, we suggest that this stress response mechanism evolved to
guard against buildup of DNA methylation errors and to coordinate
inheritance of genomic and epigenomic information.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
6 M 5-aza-CdR
(Sigma) dissolved in Me2SO. The 5-aza-CdR-containing medium
was freshly replaced every 24 h.
20 °C until use.
(growth arrest
DNA damage 45
gene) (sense,
5'-GTGTACGAGTCGGCCAAGTT-3'; antisense,
5'-AGGAGACAATGCAGGTCTCG-3'); ATF-3 (sense, 5'-AAGAGCTGAGGTTTGCCATC-3';
antisense, 5'-GACAGCTCTCCAATGGCTTC-3'); JunB (sense,
5'-TGGAACAGCCCTTCTACCAC-3'; antisense, 5'-GGAGTAGCTGCTGAGGTTGGT-3');
-actin (sense, 5'-GTTGCTAGCCAGGCTGTGCT-3'; antisense,
5'-CGGATGTCCACGTCACACTT-3'); MAGEB2 (sense, 5'-AGCGAGTGTAGGGGGTGCG-3';
antisense, 5'-TGAGGCCCTCAGAGGCTTTC-3'); BCL2-interacting killer (BIK)
(sense, 5'-GGCCTGCTGCTGTTATCTTT-3'; antisense,
5'-CCAGTAGATTCTTTGCCGAG-3'); SSX2 (sense, 5'-CAGAGTACGCACGGTCTGAT-3'; antisense, 5'-GATTCCCACGGTTAGGGTCA-3'). Amplifications were
performed in a Biometra T3 thermocycler (Biomedizinische Analytik GmbH) using the following programs: for GADD45
, first cycle 94 °C for 3 min, 58 °C for 1 min, and 72 °C for 1 min, second cycle 94 °C for 1 min, 56 °C for 1 min, and 72 °C for 1 min followed by 37 cycles of 94 °C for 1 min, 54 °C for 1 min, and 72 °C for 1 min; for ATF-3 and JunB, an initial cycle of 94 °C for 3 min
60 °C for 1 min and 72 °C for 1 min, followed by 34 cycles of
94 °C for 1 min, 58 °C for 1 min, and 72 °C for 1 min; for
-actin, first cycle 94 °C for 3 min, 64 °C for 1 min, and
72 °C for 1 min, second cycle 94 °C for 1 min, 62 °C for 1 min, and 72 °C for 1 min, followed by 25 cycles of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min; for MAGEB2, BIK, and
SSX2, first cycle 94 °C for 30 s, 62 °C for 30 s,
72 °C for 30 s, second cycle 94 °C for 30 s, 60 °C
for 30 s, 72 °C for 30 s, and 30 cycles of 94 °C for
30 s, 58 °C for 30 s, 72 °C for 30 s. The numbers
of cycles were selected and tested so that the PCR amplification remained in the linear phase. 10 µl of the PCR products were applied on a 1.2% agarose gel and visualized by ethidium bromide staining. Densitometric analysis was performed using Scion Imaging Software (Scion Inc., Frederick, MD).
20 °C for at least 30 min. After two washes with PBST-BSA, the
cells were treated with 2 N HCl at 37 °C for 30 min and
were then neutralized with 0.1 M sodium borate (pH 8.5). The cells were blocked with 10% donkey serum in PBST-BSA for 20 min at
37 °C, incubated with anti-5-methylcytidine antibody (1 µg/ml) for
45 min at 37 °C, followed by staining with donkey anti-mouse IgG
conjugated with Rhodamine Red-X (Jackson ImmunoResearch Laboratories). Finally, the cells were washed with PBS three times and were
resuspended in PBS for flow cytometry analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (24K):
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Fig. 1.
Time- and dose-dependent
inhibition of DNMT1 mRNA and DNA methyltransferase
activity by DNMT1 antisense oligonucleotide MG88. A,
the sequence of DNMT1 antisense oligonucleotide MG88 and its
mismatch control oligonucleotide MG208 are aligned to the human and
mouse DNMT1 mRNA sequence. Mismatches with the human
DNMT1 mRNA are in boldface italic type. B,
A549 cells were incubated with either 100 or 200 nM MG88 or
MG208 for either 48 or 72 h. DNA methyltransferase activity was
determined for 3 µg of nuclear extracts using a hemimethylated
substrate and 3H-labeled S-adenosylmethionine as
a methyl donor. The results presented are an average of three
determinations ± S.E. of triplicate determinations from three
independent experiments. C and D, representative
experiments from five similar experiments using competitive RT-PCR for
quantification of DNMT1 mRNA. 2 µg of RNA isolated
from A549 cells treated with either MG88 or MG208 at 200 nM
for 48 h was reverse transcribed into cDNA in the presence of
[35S]dCTP. Equal counts of labeled cDNA
(target) and increasing amounts of competitor molecules
(from 10 16 to 10
12 M) were used
as templates for PCRs with primers targeted to DNMT1
mRNA sequence. The PCR products were run on 1.2% agarose gel and
quantified by densitometry, the logarithm of the ratio of target to
competitor products were plotted against the
log of competitor
concentration (D). E, Western blot analysis of
DNMT1 expression in nuclear extracts prepared from either MG88- or
MG208-treated A549 cells (200 nM for 48 h). In the
right panel, the membrane was stained with Amido
Black to visualize the total protein transferred onto the
membrane.
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Fig. 2.
MG88 knockdown of DNMT1 inhibits DNA
synthesis and reduces the fraction of cells that synthesize DNA in A549
cells. A, A549 cells were treated with MG208 and MG88
at 200 nM for the indicated time intervals.
[3H]thymidine incorporation into DNA was quantified as
described under "Materials and Methods." The results presented are
mean ± S.D. of triplicate determinations from one of three
independent experiments that resulted in similar results. B,
Y1 cells were treated with either MG208 or MG88 at 200 nM
for 48 h. [3H]thymidine incorporation into DNA was
quantified as in A. C, A549 cells were incubated
with either MG208 or MG88 at 200 nM for 48 h. In the
last 2 h of the experiment, the cells were pulsed with 10 µM BrdUrd and were stained with anti-BrdUrd Ab and sorted
by flow cytometry. The left peak represents cells
that are negative for anti-BrdUrd staining, and the right peak
(M1) represents cells that are positive for anti-BrdUrd
staining. Similar profiles were obtained for three other independent
experiments.
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Fig. 3.
Knockdown of DNMT1 triggers
intra-S-phase arrest of DNA replication. A, A549 cells were
treated with 200 nM of either MG208 or MG88 for 48 h.
Cells pulsed with BrdUrd (10 µM) for 2 h were
stained with anti-BrdUrd antibody and propidium iodide and then
analyzed with flow cytometry. The profiles of BrdUrd staining
fluorescence versus propidium iodide staining fluorescence
are depicted. The dots representing population of cells in
the different phases of the cell cycle are color-coded
(green, G0/G1; pink,
S-phase with BrdUrd incorporation; orange, S phase
without BrdUrd incorporation; blue, G2/M). A
representative experiment of five independent experiments is presented.
B, A549 cells treated with either MG88 or MG208 for 7 days
were pulsed for 2 h with BrdUrd, stained with anti BrdUrd
antibodies and propidium iodide, and subjected to flow cytometry. The
profiles of BrdUrd staining fluorescence versus propidium
iodide staining fluorescence are depicted.
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Fig. 4.
The effects of 5-aza-CdR on DNA replication
and cell cycle kinetics in A549 cells. A, A549 cells
were treated with 1µM 5-aza-CdR (solid
bar) and the vehicle control Me2SO
(DMSO; open bar) for 24-96 h.
[3H]Thymidine incorporation into DNA was quantified as
described under "Materials and Methods." The results presented are
mean ± S.D. of triplicate determinations from one of three
independent experiments with similar results. B, A549 cells
treated with either 1 µM 5-aza-CdR or Me2SO
for 48 h were pulsed with BrdUrd (10 µM) for 2 h and stained with both anti-BrdUrd Ab and propidium iodide and then
analyzed with flow cytometry. The profiles of BrdUrd staining
fluorescence versus propidium iodide staining fluorescence
are depicted. Different cell populations are color-coded as
in Fig. 3. This experiment is a representative of three similar
experiments.
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Fig. 5.
The state of methylation of A549 cells
following DNMT1 antisense or 5-aza-CdR treatment.
A, A549 cells were treated with a 200 nM
concentration of either MG208 (red) or MG88
(green) for 48 h. Cells were harvested, stained with
anti-5-mC antibody and anti-mouse IgG conjugated with Rhodamine Red-X,
and subjected to flow cytometry. Cells stained with secondary Ab only
are used as a blank control (black line).
B, A549 cells were treated with either Me2SO
(red) or 5-aza-CdR (green) for 48 h and
subjected to flow cytometry as in A.
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Fig. 6.
Time course of global and p16 DNA
demethylation in T24 cells treated with either DNMT1 antisense or
5-aza-CdR. A, DNMT1 antisense treatment causes an
intra-S-phase arrest in T24 cells. T24 cells were treated with a 200 nM concentration of either MG208 or MG88,
Me2SO, or 1 µM 5-aza-CdR for 48 h and
were then pulsed for 2 h with BrdUrd. The cells were stained with
anti-BrdUrd Ab and propidium iodide and subjected to flow cytometry.
The profile of BrdUrd staining versus propidium iodide
staining is shown for each of the treatment groups as described in the
legend to Fig. 3. B, T24 cells were treated with 200 nM of either MG208 (red) or MG88
(green) for 48 h. Cells were harvested, stained with
anti-5-mC Ab and anti-mouse IgG conjugated with Rhodamine Red-X and
subjected to flow cytometry. Cells stained only with secondary antibody
are used as a blank control (black line).
C, T24 cells were treated with either Me2SO
(DMSO; red) or 5-aza-CdR (green) for
48 h and subjected to flow cytometry as in B. T24 cells
were treated for 48 h with a 200 nM concentration of
either MG208 or MG88 (D) or 1 µM 5-aza-C or
Me2SO (E). DNA was extracted and treated with
sodium bisulfite. Two-stage PCR was performed as described under
"Materials and Methods." The PCR products were separated on 2%
agarose gel. One of two independent experiments with identical results
is shown. L, ladder; 208, MG208-treated cells;
88, MG88-treated cells; Con,
Me2SO-treated cells; Aza, 5-aza-CdR-treated
cells; U, unmethylated; M, methylated.
2 and genes involved in interferon
response such as interferon
2, as well as the apoptosis
promoter, the BIK. BIK was previously shown to exhibit potent antitumor
activity (40) and is induced by 5-aza-CdR and sodium butyrate in
hepatic cancer cell lines (41). The induction of the interferon
response pathway was previously proposed to be a major cellular
response to 5-aza-CdR (42). However, in addition to up-regulation of genes that are potentially antimitotic and proapoptotic, the most remarkable induction occurred in three groups of cancer/testis-specific genes residing on the X chromosome, which are exclusively expressed in
testis and a wide variety of tumors but not in nontumor tissues. These
are the GAGE (G antigen 7) family (43), the MAGE
(44-48) family of melanoma antigens, and the genes residing at the
synovial sarcoma X breakpoint, SSX2-4 (49, 50). It is well
documented that the MAGE (44-46) and GAGE family
of genes as well as SSX2 are controlled by DNA methylation
and are induced by DNA-demethylating agents (51).
Genes up-regulated by 5-aza-CdR after 48 h of treatment
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Fig. 7.
5-aza-CdR treatment induces expression of the
proapoptotic gene BIK and cancer/testis-specific genes
MAGEB2 and SSX2. A,
total RNA was isolated from A549 cells treated with either 5-aza-CdR (1 µM) or Me2SO (DMSO) as a control
for 24-96 h. RT-PCR was performed with primers for the indicated genes
as described under "Materials and Methods." 10 µl of PCR products
were run on 1.2% agarose gel. B, PCR products were
quantified by densitometry, normalized to -actin, and presented as
arbitrary units for SSX2 and MAGEB2 or as a ratio
of 5-aza-CdR (Aza) to Me2SO for BIK,
ATF-3, GADD45, and JunB.
Representative data from two separate experiments are shown.
, and JunB. These three genes were not
found to be induced by 5-aza-CdR treatment in the gene array analysis,
and this result was confirmed by RT-PCR (Fig. 7). Their induction
profiles following antisense treatment were verified using
semiquantitative RT-PCR. The induction peaked at 48 h for
GADD45
and JunB and at 72 h for
ATF-3. This profile of induction is consistent with the
hypothesis that the cell recognizes DNMT1 knockdown as a genotoxic
challenge and reacts by inducing a stress response gene expression
program. The kinetics of this response, early induction followed by
mitigation of the response (Fig. 8), is inconsistent with the mechanism
involving passive demethylation of DNA. Consistent with this hypothesis that MG88 action at 48 h is independent of DNA methylation is the
fact that the genes induced by the DNA-demethylating agent 5-aza-CdR
(MAGEB2 and SSX2) were not induced by MG88 treatment for
48 h (Fig. 8).
Stress-responsive genes up-regulated by MG88 after 48 h of
treatment
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Fig. 8.
DNMT1 antisense knockdown induces
expression of the genotoxic stress response genes
ATF-3, JunB, and
GADD45 . A, total RNA was
isolated from A549 cells treated with 200 nM of either
MG208 or MG88 for 24-96 h. RT-PCR was performed with primers for the
indicated genes as described under "Materials and Methods." 10 µl
of PCR products were run on 1.2% agarose gel. In the case of
MAGEB2, SSX2, and BIK, RT-PCR was also performed
on A549 cells treated with 5-aza-CdR (1 µM) for 72 h
as an expression control. B, PCR products were quantified by
densitometry and normalized to
-actin product. The relative ratio of
MG88-treated to MG208-treated cells were then calculated and presented
in B. Representative data from 2-4 separate experiments are
shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
. The most established function of p21 is at the
G1/S boundary (62), and GADD45
has been shown to play an
important role in the G2/M checkpoint in response to DNA
damage (63), but their involvement in the intra-S-phase checkpoint is
unclear. However, it is possible that inhibition of Cdk2 by p21 can
lead to S-phase arrest. An additional possibility is that the assembly
of the replication fork requires the presence of DNMT1 and that its
absence from the fork is what signals arrest of DNA replication.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Hudson and the Montreal Genome Center for support and help in performing and analyzing the microarrays. We thank Gula Sadvakassova for technical assistance and Nancy Detich for critical review of the manuscript.
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FOOTNOTES |
---|
* This work was supported by the Canadian Institute of Health Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These two authors contributed equally to this work.
To whom correspondence should be addressed: Dept. of
Pharmacology and Therapeutics, McGill University, 3655 Sir William
Osler Promenade, Montreal, Quebec H3G 1Y6, Canada. Tel.:
514-398-7107; Fax: 514-398-6690.
Published, JBC Papers in Press, February 7, 2003, DOI 10.1074/jbc.M213219200
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ABBREVIATIONS |
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The abbreviations used are: 5-aza-CdR, 5-aza-2'-deoxycytidine; PBS, phosphate-buffered saline; BSA, bovine serum albumin; BrdUrd, bromodeoxyuridine; BIK, BCL2-interacting killer; Ab, antibody; RT, reverse transcriptase.
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