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INTRODUCTION |
Most eukaryotic cells do not divide indefinitely. This phenomenon
of limited proliferative capacity, termed replicative senescence, was
initially documented in cultures of human fibroblasts (1). Since then,
it has been extended to a number of cell types and species and has
become a model for aging at the cellular level. Replicative senescence
is characterized by an irreversible growth arrest that is the
consequence of repeated cell division. The number of divisions that
normal cells complete before they enter senescence is constant under
standard conditions and depends on the species, cell type, age, and
genetic background of the donor (2). This suggests the existence of a
mechanism by which cells sense the number of divisions completed.
Telomere erosion, resulting from the incapacity of DNA polymerase
to replicate the end of linear chromosomes, has emerged as a major
candidate for the counting mechanism of human cells (3). The telomere
hypothesis of cellular aging has been greatly strengthened by
experiments demonstrating that ectopic expression of the catalytic
subunit of telomerase (hTERT)1 before senescence
prevents telomere shortening and allows indefinite cell proliferation
(4). The expression of hTERT correlates with the presence of telomerase
activity in human cells, and the role of telomerase in immortalization
is generally believed to involve the prevention of telomere erosion.
However, it was recently reported that telomerase extends the life span
of SV40-transformed human fibroblasts without net telomere lengthening
(5) and that stable clones expressing limiting amounts of hTERT and
having shorter telomeres than the parental senescent cells continue to proliferate (6). This indicates that the resolution of the telomere
replication problem is not always necessary for extension of the life
span. In addition, the chromosomal instability that occurs when
telomeres erode sufficiently, causing a crisis like state in
pre-immortalized SV40-transformed cells, is not a characteristic of the
normal aging phenotype and occurs only when senescence has been
circumvented. Furthermore, immortal cell lines in which telomerase
was inhibited by a dominant negative form of the enzyme undergo
apoptosis instead of entering senescence (7, 8).
The gradual loss of DNA methylation has been proposed as an alternative
counting mechanism (9, 10). In human cells, 60-90% of the cytosines
in CpG dinucleotides are methylated at the carbon 5 position, leaving a
minor portion of the genome methylation free. Methylation of CpG
islands is associated with transcriptional silencing and the formation
of condensed chromatin structures enriched in hypoacetylated histones
(11, 12). DNA methylation has been shown to be essential for normal
development, X chromosome inactivation, imprinting, and has been
suggested to play a role in in vitro cellular aging. The
extent of CpG methylation decreases during serial passage of normal
cells in culture (9, 13) and during aging of organisms (14, 15). On the
other hand, most immortal cell lines maintain constant levels of
genomic DNA methylation. However, it is not known whether the
aging-related loss of methylation involves specific regions of the
genome or if it is a stochastic process.
The transfer of a methyl group from S-adenosylmethionine to
the 5 position of cytosines in CpG dinucleotides is catalyzed by the
enzyme DNA (cytosine-5) methyltransferase (DNAMeTase) (16). DNAMeTase activity has been shown to be elevated in cancer cells in vitro, in tumors in vivo (17), and in
transformation triggered by oncogenic Ras or SV40 T antigen (18,
19). In addition, increased DNAMeTase levels are required to maintain
the phenotype of fibroblasts transformed with ras and
with the fos oncogene (20), suggesting that the
maintenance of DNAMeTase activity, and hence DNA methylation levels, is
an important step in cellular transformation. Consistent with the loss
of DNA methylation, DNAMeTase activity was observed to decrease with
increasing population doublings in serially passaged normal fibroblasts
(21).
One of the major effectors of the senescence phenotype is p21. p21
mRNA and protein are significantly increased as fibroblasts enter
senescence (22). Human diploid fibroblasts in which both copies of the
gene have been inactivated by homologous recombination fail to undergo
normal senescence (23). They have an extended in vitro life
span, and rather than attaining a senescent state, they enter a
crisis-like state in which DNA synthesis and cell death occur
simultaneously. In addition, p21 appears to be needed for the induction
of the senescent-like state by many treatments such as sodium butyrate,
which induce premature growth arrest and many features of senescence
(24). Although it is known that p21 blocks the action of the
cyclin-dependent kinases (25) and prevents the
phosphorylation of Rb (26), thereby preventing the induction of many of
the genes needed to traverse the G1/S boundary (27); the
role of p21 in cellular senescence is not completely understood, and a
mechanism for the induction of p21 at the end of the in
vitro life span of a fibroblast cell culture has not yet been
convincingly demonstrated. Very recently it was reported that p21 could
be induced by inhibition of DNAMeTase (28, 29). Thus, it appears that
DNAMeTase itself, independent of DNA methylation, can inhibit gene
transcription. The exact mechanism of inhibition is not known, but
DNAMeTase does have a repressive domain, and it interacts with
repressive complexes that include histone deacetylases, which
appear to contribute to a repressive effect on the p21 promoter
(30-32). Since DNAMeTase activity decreases during cellular aging, the
repressive effect on the p21 promoter may be overcome, and the
chromatin structure surrounding the p21 promoter may be more active,
resulting in increased p21 transcription and cellular senescence.
In this report we describe the effect of DNAMeTase inhibition on normal
human diploid fibroblasts. We demonstrate that inhibition of DNAMeTase
induces a senescence-like cell cycle arrest that is mediated by and
requires functional p21. Our results support the hypothesis that
DNAMeTase activity and/or methylation levels could act as a cell
division counting mechanism in human cells.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
HCA2, human diploid fibroblasts isolated in
this laboratory from neonatal foreskin, and all human cells lines used
were maintained and made quiescent as described previously (33). Human
lung fibroblasts (LF1), and LF1 derivatives with a homozygous deletion of both p21 alleles designated H07.2-1, were a generous gift from J. Sedivy (23).
Determination of the 5mC Content--
Genomic DNA was isolated
using the DNAZol DNA extraction kit (Molecular Research Center, Inc.)
according to manufacturer's directions. RNA was removed by alkaline
hydrolysis (0.5 M NaOH, 37 °C for 1 h). The 5mC
content in DNA samples was determined by HPLC analysis of enzymatic
hydrolysates of DNA. 10 µg of DNA was digested at 37 °C for 3 h using 2 units of microccocal nuclease (United States
Biochemical, Cleveland, OH) and 2 µg of spleen phosphodiesterase II (Roche Diagnostics Corp., Indianapolis, IN) in 10 mM CaCl2 and 20 mM sodium
succinate, pH 6.0. The resulting 3'-deoxymonophosphate nucleosides were
further hydrolyzed by overnight incubation at 37 °C with 20 units of
alkaline phosphatase (Amersham Pharmacia Biotech). Samples were
injected into a Beckman Ultrasphere ODS, 4.6 mm × 25 cm (5 µm
particle size) column at room temperature, programmed as follows: 100%
buffer A (2% methanol in 0.05 M potassium phosphate, pH
4.5) for 10 min, injection of the samples, elution for 20 min in 100%
buffer A, followed by buffer B (9% methanol in 0.05 M
potassium phosphate, pH 4.5) over a 5-min linear gradient up to 100%
buffer B for 10 min. HPLC mobile phase was delivered to the column at
0.3 ml/min. Absorbance at 280 nm was recorded. The percent
methylcytosine in the genome was determined as a ratio of the area of
the 5mC peak to the total area of methylcytosine and cytosine residues
in the sample.
CdR Treatment--
Cells were plated at a density of
104 cells/cm2 and 24 h later treated with
0.5 µM 5-aza-2-deoxycytidine (CdR) (Sigma). The medium
was changed every 24 h for medium containing fresh CdR for the
time required to control cells to achieve three population doublings.
Determination of Cell Cycle Profile--
Cells (2 × 106) were fixed with 70% ethanol for 30 min, stained with
50 µg/ml propidium iodide, and treated with 10 µg/ml RNase A. DNA
content at each cell cycle stage was determined by flow cytometry.
DNA Methyltransferase Activity--
Cells were harvested by
trypsinization, washed twice in phosphate-buffered saline, and
resuspended in 0.1 ml of hypotonic lysis buffer containing 50 mM Tris-HCl, pH 7.8, 1 mM EDTA, 1 mM dithiothreitol, 0.01% NaN3, 10%
glycerol, 1% Tween 80, 60 µg/ml phenylmethanesulfonyl fluoride, and
100 µg/ml RNase A. The cells were lysed by four cycles of freezing in
dry ice-ethanol and thawed at 37 °C. Protein concentration of the
supernatant, after centrifugation of the cell lysates at 15,000 × g for 20 min, was determined by the Bradford method
(Bio-Rad). The enzyme activity was measured as described
previously (16), with slight modifications. Briefly, a 20-µl reaction
mixture containing 5 µg of cell extract protein, 0.5 µg of a
hemimethylated oligonucleotide duplex corresponding to the imprinted
locus SNRPN (small nuclear riboprotein-associated peptide N)
exon-1, and 3.3 µCi of
S-adenosyl[methyl-3H]methionine (92 Ci/mmol; Amersham Pharmacia Biotech). The mixture was incubated at
37 °C for 2 h and the reaction terminated by the addition of
300 µl of a solution containing 1% sodium dodecyl sulfate (SDS), 2 mM EDTA, 3% 4-aminosalicylate, 5% butanol, 125 mM NaCl, 0.25 mg/ml salmon sperm DNA, and 1 mg/ml
proteinase K. After incubating for a further 30 min at 37 °C, the
reaction mixture was extracted with an equal volume of
phenol/chloroform/isoamilic alcohol and ethanol-precipitated. DNA was
dissolved in 0.3 N NaOH and incubated at 37 °C for
2 h. DNA was collected on a glass fiber filter disc, saturated
with 1 mM nonlabeled AdoMet, and washed with 5%
trichloroacetic acid followed by 70% ethanol. The filter was
air-dried and the radioactivity measured by
scintillation counting
in 5 ml of scintillation fluid. After subtracting background, the
radioactivity incorporated into the DNA as a measure of DNAMeTase activity was determined. Reactions were performed in triplicate and the
results expressed as the mean ± S.D.
Oligonucleotides--
2'-O-Methylphosphorothioate
oligonucleotides were used in the antisense experiments: DNAMeTase
antisense (MG88), AAGCATGAGCACCGTTCTCC and
unrelated control oligonucleotide,
ATACAACATGACAATAGATCG (boldface nucleotides are
2'-O-methyl-modified). The following oligonucleotides were
used as substrate for the DNAMeTase activity determinations. The
hemimethylated duplex was prepared by annealing equimolar amounts of
SNRPN-meth,
CTTGCCMGCTCCATMGMGTCACTGACMGCTCCTCAGACAGATGMGTCAGGCATCTCMGGMGGCMGCTCCACTCTG (methylated single strand oligonucleotide), and SNRPS-unmeth, CTTGCCMGCTCCATMGMGTCACTGACMGCTCCTCAGACAGATGMGTCAGGCATCTCMGGMGGCMGCTCCACTCTG (unmodified single strand oligonucleotide).
The complementary oligonucleotides were incubated in annealing buffer
(50 mM Tris-HCl, pH 7.4, 1 mM
Na2EDTA, and 100 mM NaCl) at 95 °C for 5 min
followed by 65 °C for 15 min and 37 °C for 15 min (34). The
full-length duplexes were purified on a 15% polyacrylamide gel,
dissolved in TE (10 mM Tris-HCl, pH 7.8, 1 mM EDTA), and stored at 20 °C.
Oligonucleotide Treatment--
Cells (5 × 105)
were electroporated on three sequential days (300 V, 960 microfarads) with a 100 nM concentration of
the hybrid 2'-O-methylphosphorothioate DNAMeTase antisense or
unrelated oligonucleotide in freshly made cytomix buffer (120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4, 10 mM
KH2PO4, 25 mM Hepes, 2 mM EGTA, 5 mM MgCl2, 2 mM ATP, 5 mM glutathione, pH 7.6). After each
electroporation the cells were incubated in complete medium for 48 h in the presence of 40 nM oligonucleotide.
Transient Transfection of an Antisense p21 Expression
Vector--
The CMV-AS-p21 plasmid contained 1-165 nucleotides of p21
cDNA in the opposite orientation. CMV control vector (empty vector) and CMV-AS-p21 were cotransfected with the CMV-EGFP plasmid into HCA2
cells (PD 19) using LipofectAMINE Plus (Life Technologies, Inc.).
Transfection medium (0% fetal bovine serum) was replaced with 0.5 µM CdR, 10% fetal bovine serum medium 3 h after
plasmid delivery.
SA-
-galactosidase Activity--
Staining for
SA-
-galactosidase (
Gal) activity was performed as described
previously (35).
[3H]Thymidine Labeling--
Thymidine
incorporation was performed as described previously (33).
SV40 T Antigen Electroporation--
PSV7, an SV40 early
promoter-driven T antigen expression vector (10 µg), was introduced
into HCA2 cells by electroporation (300 V, 960 microfarads)
using 0.5 × 106 cells in 400 µl of freshly made
cytomix buffer.
Determination of the Colony Size Distribution--
Cells were
plated at a density of 20 cells/60-mm dish and incubated undisturbed
for 2 weeks in 10% fetal bovine serum. Dishes were then fixed for 5 min in 1% glutaraldehyde, washed gently with water, and stained with
1% crystal violet. The number of cells in the individual colonies was
determined using a dissecting microscope.
Northern Blot Analysis--
Northern analysis was performed
according to standard procedures. 32P-Labeled DNA probes
for human mRNAs were prepared by random priming.
Western Blot Analysis--
Samples containing equal amounts of
protein (20 µg) from cell lysates were separated by
SDS-polyacrylamide gel electrophoresis and then transferred to a nylon
membrane. Proteins were detected by incubation with the indicated
antibodies and enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech).
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RESULTS |
Total 5-Methylcytosine Content in Normal Diploid Fibroblasts Is
Reduced during Cellular Aging--
The normal human diploid fibroblast
cell line HCA2 can replicate in culture for 80-90 population doublings
(PD). To determine the rate of reduction in total DNA methylation
during in vitro aging in HCA2 cells, we measured
5-methylcytosine (5-mC) content in genomic DNA from cells at increasing
PD. Reverse phase HPLC revealed that 5mC content was reduced from 5.3%
(±1.03) to 2.2% (±0.61) from PD 27 to PD 80 in culture (Fig.
1A). The decrease in DNA
methylation levels during in vitro aging was accompanied by
a decrease in DNAMeTase activity (Fig. 1B). The tritiated
thymidine labeling index of the above cultures indicated that the
decrease in DNAMeTase activity was not related to the percentage of
proliferating cells, but rather related to the proliferative capacity
of the culture.

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Fig. 1.
Global DNA methylation levels and DNAMeTase
activity decrease as cells age in culture and in CdR-treated
cells. A, genomic methylation levels were measured in
HCA2 cells at the indicated PD by reverse phase HPLC at a pH of 4.5. The percentage of methylated cytosine was determined after enzymatic
hydrolysis. In the case of CdR-treated cells (PD 28 CdR, solid
bar), 0.5 µM CdR was added to the culture medium
every day for 5 days and the cells harvested 48 h after the last
CdR addition. B, DNAMeTase activity was measured at the
indicated PD using an hemimethylated substrate as described under
"Experimental Procedures." The percentage of dividing cells,
analyzed by [3H]thymidine incorporation (48 h) is
indicated inside the bars.
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DNA methylation levels can be manipulated by inhibition of the DNA
(cytosine) methyltransferase (DNAMeTase) (36). To induce a premature
decrease in global DNA methylation levels, we treated HCA2 cells with
CdR, a potent DNAMeTase inhibitor. HPLC analysis indicated that HCA2
cells (PD 28) incubated for 5 days (2-3 doublings) with 0.5 µM CdR (see "Experimental Procedures") exhibited
rapid demethylation, and levels decreased to those observed in
senescent cells (Fig. 1A).
DNAMeTase Inhibition Induces Growth Arrest in HCA2 Cells--
We
analyzed the phenotype of the CdR-treated cells by measuring tritiated
thymidine incorporation and cell number in the culture. Cell counts at
different times during and after the CdR treatment indicated that the
demethylation was accompanied by cessation on culture growth (Fig.
2A). Quantitation of labeled
nuclei in HCA2 cells, incubated for 48 h in the presence of
tritiated thymidine at the end of the treatment, confirmed this
observation and suggested that the cells withdraw from the cell cycle
(Fig. 2B). The cell cycle profile of the treated cells by
flow cytometry demonstrated that the CdR-treated cells were arrested
both in G1 and G2, resembling normal
fibroblasts in late senescence (Table
I).

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Fig. 2.
DNAMeTase inhibition induces cell cycle
arrest. A, the effect of 0.5 µM CdR
treatment on the growth of young human fibroblasts. HCA2 cells (PD 25)
were seeded at a density of 104 cells/cm2 and
split at the same density every 3 days. An aliquot of cells was counted
at each split to compute the PD achieved. B, cells were
cultured for 24 h in the presence of tritiated thymidine
([3H]TdR) 3 days after the last addition of CdR. The
percentage of labeled nuclei before (white bars) and
after (solid bars) CdR treatment was scored microscopically.
Control cultures had been treated the same way as CdR cultures, but no
drug was added to the medium.
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Table I
The decline in growth rate was coupled to an increase in the proportion
of cells in the G2 phase of the cell cycle
FACS analysis (propidium iodide cytofluorometry), followed by computer
determination of the percentage of cells in the different phases of the
cell cycle, were carried out on control cells at 25 and 80 PD and on
CdR (0.5 µM) treated cells at 25 PD.
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The loss of proliferative capacity induced by the DNAMeTase inhibitor
was irreversible, as the cells did not re-enter the cell cycle for at
least up to 3 weeks after removal of the drug. Treatment of normal
young cells made quiescent by serum starvation with the cytosine
analogue did not affect the ability of the cells to respond to serum
stimulation (data not shown). This demonstrates that incorporation of
CdR into DNA is necessary for DNAMeTase inhibition and induction of
growth arrest.
To determine the specificity of the response, we exposed the cells to
DNAMeTase antisense oligonucleotides (28). We electroporated HCA2 cells
with 100 nM antisense oligonucleotides every other day for
5 days. Four days after the final electroporation we plated them at a
density of 20 cells/60-mm dish. This treatment resulted in a decrease
in the colony size distribution, a measure of the growth ability that
was not observed in cells treated with an unrelated
phosphorothioate oligonucleotide (Fig.
3). This indicates that the growth arrest
is not due to pleiotropic effects of the CdR inhibitor but is rather
induced by inhibition of DNAMeTase.

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Fig. 3.
Treatment of normal fibroblasts with
DNAMeTase antisense oligonucleotide induces cell cycle arrest.
HCA2 cells were electroporated with MG88, a DNAMeTase antisense
oligonucletide (solid bars), or with an unrelated
oligonucleotide (white bars) and split at a density
of 20 cells/60-mm dish. Two weeks later the colonies were fixed and
stained with crystal violet, and the number of cells per individual
colony was determined by bright field microscopy.
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DNAMeTase Inhibition-induced Growth Arrest Has Many Senescent
Cell-like Features--
To further characterize the CdR-induced
proliferative arrest we tested some of the known senescence-associated
changes in the arrested cells. CdR-treated cells acquired a
senescent-like morphology, increased size, and expressed the
senescence-associated
-galactosidase activity (Fig.
4A). Northern analysis of the
expression of type I collagenase, a gene known to be overexpressed in
senescence, revealed that collagenase expression was induced in HCA2
cells after CdR treatment (Fig. 4B).

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Fig. 4.
CdR treatment induces a senescent-like
morphology and expression of senescent-associated proteins.
A, photomicrographs of HCA2 control and 0.5 µM
CdR-treated cells stained for -galactosidase activity at pH 6 (35).
B, HCA2 cells were seeded at standard density
(104/cm2) and 24 h later treated with 0.5 µM CdR for 7 days. Steady state mRNA levels of
collagenase were examined by Northern blot analysis. Probing the filter
for -actin was used to determine equal loading.
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The hallmark of cellular senescence is the inability of senescent cells
to be stimulated to synthesize DNA by the addition of mitogens or
serum. The only agent that overcomes this terminal arrest is SV40 T
antigen (37). To test the ability of CdR-arrested cells to re-enter the
cycle upon T antigen introduction, we analyzed tritiated thymidine
incorporation of cells in which the T antigen expression plasmid (PSV7)
had been introduced. HCA2 senescent (PD 88) and CdR-treated cells (9 days post-treatment) were electroporated with the PSV7 plasmid and
24 h later incubated for 48 h in the presence of tritiated
thymidine. We quantitated the number of cells that expressed T antigen
(positive for immunocytochemistry using an anti-T antigen antibody),
the number that synthesized DNA (TdR-labeled nuclei), and the number
that exhibited both features. T antigen expression induced both
senescent and CdR-arrested HCA2 cells to synthesize DNA (Fig.
5). As expected, untransfected cells (not
stained by the antibody) remained arrested (Fig. 5).

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Fig. 5.
The block in DNA synthesis induced by
DNAMeTase inhibition can be reversed by SV40 large T antigen.
CdR-treated (PD 25) and senescent HCA2 cells were electroporated with
pSV7 and maintained for 24 h in medium containing
[3H]thymidine. Cells were fixed and processed for the
simultaneous detection of T antigen immunoreactivity and thymidine
incorporation. White bars, percentage of labeled
nuclei in cells not expressing T antigen. Solid bars,
percentage of labeled nuclei in cells expressing T antigen.
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P21 Is Required for the Growth Arrest Mediated by Inhibition of
DNAMeTase--
We further characterized the arrested phenotype by
analyzing the pattern of expression of cell cycle proteins. Western
analysis of CdR-treated HCA2 cells showed no changes in p53, p33, p27, or p16 immunoreactivity (Fig.
6A). However, there was a
5-6-fold increase in p21 protein levels following CdR treatment (Fig.
6A). Northern analysis indicated that the increase in p21
protein was accompanied by an elevation of p21 mRNA levels (Fig.
6C). The increase in p21 mRNA was evident after 1 population doubling in the presence of the drug (Fig. 6C,
lane 2). p21 protein was elevated after 2 PD and
continued to increase until the end of the treatment (Fig.
6B).

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Fig. 6.
CdR treatment induces changes in p21 mRNA
and protein levels. A, protein extracts (20 µg)
obtained from control (lanes 1 and 3) or treated
with 0.5 µM CdR (lanes 2 and 4),
HCA2 cells (PD 27) (lanes 1 and 2), and O41
(lanes 3 and 4) cells were subjected to
Western analysis for the immunodetection of p53, p33, P21, p16, and
actin as loading control. B, cells were harvested at the
indicated times (days) after the first addition of 0.5 µM
CdR, and p21 immunoreactivity was analyzed by Western blot.
C, total RNA from cells treated for the indicated time (1-5
days after the first addition of the drug) with 0.5 µM
CdR was subjected to Northern blot analysis for the determination of
p21 mRNA levels.
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To identify the molecules involved in the cell cycle arrest caused by
inhibition of DNAMeTase, we treated a series of cell lines bearing
mutations in known cell cycle regulators. These included cells lacking
a functional p53 (T98G, Susm1, EJ, SaOS-2, 041), Rb (HeLa, Susm-1,
SaOS-2, J82,), and p16 (T98G, 041, HT1080, MCF7, HCT116). 0.5 µM CdR treatment for 2-3 PD in culture induced cell
cycle arrest in all the cell lines analyzed independent of the
mutation, indicating that these proteins are not required for the
CdR-induced senescent-like state (data not shown). Nevertheless, a
subset of these cell lines that includes CMV, EJ, O41, and HCT116 cells
exhibited no p21 induction after the treatment. These cell lines have
been reported to have the p16 promoter hypermethylated, and therefore
expression of the gene is repressed. As expected, they displayed a very
strong re-expression of p16 upon treatment with CdR (Fig.
6A).
We next examined the significance of the p21 gene in the arrest induced
by inhibition of DNAMeTase. For this purpose, we used the parental
normal human diploid lung fibroblast cell line LF1, along with H07.2-1
cells, in which the p21 gene has been deleted by double homologous
recombination. CdR treatment induced LF1 to enter a senescent-like
state, with kinetics similar to that observed for HCA2 (normal
fibroblasts derived from neonatal foreskin). These cells also showed an
early induction of p21 mRNA followed by an increase in p21 protein
levels (data not shown). In contrast, there was no effect of the CdR
treatment on the cell cycle of the H07.2-1 cells, measured by
[3H]TdR incorporation (Fig.
7) and cell number (data not shown). To
rule out the possibility of differential incorporation of the drug into
the DNA, or altered DNAMeTase response in the
p21
/
cells, we measured genomic DNA
methylation levels in the CdR-treated cells. HPLC analysis indicated
that inhibition of DNAMeTase by CdR treatment decreased global
methylation levels in H07.2-1 cells from 2.46 ± 0.42% to
1.8 ± 0.4% (Fig. 8). Taken
together, these data suggest that that the DNAMeTase inhibition-induced
arrest is mediated by p21 and is not dependent on p53, Rb, or p16.

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Fig. 7.
DNAMeTase inhibition-induced cell cycle
arrest is dependent on the presence of p21. HO7.2-1
(p21 / ) cells were split at a density of
104 cells/cm2 24 h before the addition of
0.5 µM CdR. Media were changed every day for media
containing fresh CdR for 14 days. [3H]Thymidine
incorporation was measured for 24 h at the end of the treatment.
White bars indicate the percentage of labeled nuclei
before the addition of CdR. Solid bars indicate the
percentage of labeled nuclei observed at the end of CdR treatment.
Control cultures had been treated the same way as CdR cultures, but no
drug was added to the medium.
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Fig. 8.
CdR treatment induces demethylation in
p21 /
cells. Genomic DNA from young and senescent p21+/+
(LF1, white bars) and
p21 / (HO7.2-1, solid bars) cells
control (control) and CdR treated (CdR) cells was subjected to HPLC
analysis for the determination of DNA methylation levels.
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Interestingly, the 5mC content of the H07.2-1 cells was lower than that
of senescent cells (Fig. 8). These cells had bypassed senescence and
were in an extended life span period (23). The decrease in methylation
levels relative to the parental cells and to levels below that of
senescent fibroblasts suggest that the subcultivation-related
demethylation persists in cells that have circumvented senescence.
Expression of Antisense p21 mRNA Eliminates DNAMeTase
Inhibition-mediated Growth Arrest--
The causal association between
p21 and the DNAMeTase inhibition-induced arrest was further
corroborated in HCA2 cells. We determined the effect of antisense p21
mRNA expression on the ability of DNAMeTase inhibition to halt
proliferation in HCA2 cells. Expression of antisense p21 mRNA
decreased p21 protein to undetectable levels (Fig.
9A) and has been shown to
abolish p21-mediated growth arrest in cotransfection assays (38).
Cotransfection of CMV-AS-p21, expressing antisense p21 mRNA, +1 to
+165, with CMV-EGFP into HCA2 cells was followed by addition of CdR or
control medium 3 h after plasmid delivery. CdR treatment was
continued for 7 days. Expectedly, cells receiving control DNA (empty
vector) exhibited a marked decrease in DNA synthesis, assayed as
thymidine incorporation. 96% of CdR-treated cells failed to
incorporate tritiated thymidine (data not shown), regardless of GFP
expression. In contrast, the expression of antisense p21 mRNA
abolished CdR-induced growth arrest. 85% of GFP expressing cells
(presumably expressing antisense p21 mRNA) exhibited thymidine
incorporation (Fig. 9B). GFP-positive cells that are still
responsive to CdR treatment (15%) may represent a subpopulation of
cells transfected only with the GFP construct, or that express low
levels of antisense p21 mRNA. On the other hand, only 3% of cells
negative for GFP expression synthesized DNA (Fig. 9B). These
data support the conclusion that DNAMeTase inhibition induces
growth arrest through the p21 protein.

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Fig. 9.
p21 protein is responsible for CdR inhibition
of DNA synthesis. A, expression of antisense p21
mRNA decreases p21 protein levels. Increasing amounts (0.0-1.5
µg, as indicated) of the CMV-AS-p21 construct (expressing antisense
p21 mRNA) was cotransfected with 0.25 µg of CMV-p21 (expressing
p21) into HCA2 cells, and 48 h later cells were harvested and
subjected to Western analysis for the immunodetection of p21 and actin.
B, expression of antisense p21 mRNA blocks CdR-induced
DNA synthesis inhibition. HCA2 cells (PD 19) were cotransfected with
CMV-EGFP (0.7 µg) and CMV-AS-p21 (1.5 µg) plasmids. Transfection
medium was replaced 3 h after plasmid delivery with medium
containing 0.5 µM CdR. Media were changed every day for
media containing fresh CdR for 7 days. [3H]Thymidine
incorporation was measured for 24 h at the end of the treatment.
Photomicrographs of two representative fields under UV light
(left panel, taken before autoradiographic development),
phase contrast and bright field superimposed (middle panel),
and bright field (right panel). Black arrows in
the middle panel indicate untransfected cells (GFP-negative)
arrested by the CdR treatment, black arrowheads denote
transfected cells (GFP-positive) with labeled nuclei, and white
arrowheads indicate transfected cells (GFP-positive) that are
nevertheless arrested.
|
|
 |
DISCUSSION |
The results presented here indicate that inhibition of DNAMeTase
in normal diploid human fibroblasts, as well as in a variety of human
immortal cell lines, results in an irreversible arrest in cell
proliferation. The arrest occurred when either CdR or DNAMeTase
antisense oligonucleotides were used, indicating that the inhibition of
replication is not a consequence of the pleiotropic effects of CdR. The
arrested phenotype exhibited many characteristics of a senescent-like
state, including cell morphology, DNA content, expression of the
SA-
galactosidase activity, induction of type I collagenase, and
p21 expression. In addition, the DNA synthesis inhibition could be
overcome by expression of the SV40 T antigen, a hallmark of senescent
cells. The data indicate that DNAMeTase inhibition causes growth arrest
in normal human fibroblasts by up-regulation of the cell cycle
inhibitor p21. p21 induction appears to be essential for the growth
arrest to occur, as p21 minus cells and cells expressing antisense p21
mRNA are refractory to DNAMeTase inhibition. The lack of induction
of SA-
-galactosidase activity and type I collagenase expression in
p21
/
CdR-treated cells suggests that these
are downstream effects of p21 induction. Alternatively, they could be
manifestations of the arrested phenotype. We favor the latter
possibility as cells arrested by serum starvation express high levels
of p21 but do not exhibit any of these markers. A similar absolute
requirement of p21 for growth arrest has been shown in response to
other stimuli, including histone deacetylase inhibition,
-irradiation, and BRCA1 overexpression (39-41). Conversely, p21 is
not essential in the growth arrest induced by serum starvation (our
data and Ref. 39).
Levels of the tumor suppressor p53 were not modified by DNAMeTase
inhibition, and p21 up-regulation and cell cycle arrest after DNAMeTase
inhibition were also observed in cell lines lacking a functional p53.
This suggests that p21 up-regulation in this case is most likely to be
independent of p53. This further supports the idea that p21 is
up-regulated upon DNAMeTase inhibition by a p53-independent mechanism.
Recently, it has been reported that inhibition of DNAMeTase by
DNAMeTase antagonists or antisense oligonucleotides results in p21
induction with a concomitant inhibition of DNA replication (28, 29).
Our results suggest that transcriptional activation accounts for p21
induction after DNAMeTase inhibition as p21 mRNA levels are rapidly
increased after treatment. The demonstration that DNAMeTase has a
transcriptional repression domain and associates with histone
deacetylase activity and the corepressor DMAP1 suggests that p21
transcriptional activation resulting from DNAMeTase inhibition involves
derepression of the p21 promoter (30, 32). In addition, p21 expression
is induced by histone deacetylase inhibitors through a process
involving histone hyperacetylation (42). Thus, the molecular mechanism for DNAMeTase repression of p21 could involve recruitment of histone deacetylase activity to the p21 promoter.
The observed proliferation inhibition was also independent of Rb and
p16 status, as cell lines deficient in these proteins exhibited cell
cycle withdrawal and p21 induction after DNAMeTase inhibition. This
suggests that DNAMeTase repression of p21 is not mediated by
interaction with the repressive DNAMeTase-Rb-E2F1 complex (31).
Despite their similar phenotype after CdR treatment, cell lines that
had p16 inactivated by methylation showed a different pattern of
immunoreactivity of cell cycle regulatory proteins in response to
DNAMeTase inhibition from cell lines in which p16 was not methylated.
Western analysis indicated that cells reported to have the p16 promoter
methylated (CMV, EJ, O41, HCT116), and therefore repressed, produced
high levels of the protein after CdR treatment, suggesting promoter
demethylation. The level of p16 immunoreactivity present in these cells
after demethylation was much higher than that observed in other cell
lines, normal senescent cells or p21
/
cells
that had overcome senescence and thus had high p16 levels. This
observation suggests that methylation of the p16 promoter introduces a
modification of chromatin structure that not only limits accessibility
of transcriptional activators, but also prevents basal repressors from
acting on the p16 gene. The relief of these constraints by inactivation
of DNAMeTase allows the transcriptional machinery to gain preferential
access to the promoter region, leading to disregulated gene expression.
High expression of p16 per se could induce the observed
growth arrest in this subset of immortal cell lines with p16 promoter hypermethylated.
The incorporation of the CdR base analogue into the genomic DNA traps
DNAMeTase covalently, resulting in inhibition of its activity.
Therefore, the observed arrest could be attributed to an interference
of this adduct in the DNA replication process. However, this
possibility seems to be ruled out by the T antigen induced DNA
synthesis data and the observation that cells lacking the p21 protein
(p21
/
or antisense p21 expressing cells)
can proliferate normally in the presence of the drug.
Several lines of evidence now indicate that normal cells have a
mechanism that permits the counting of cell divisions. Alteration of
such a mechanism is important and necessary for immortalization. The
data presented here suggest that DNAMeTase activity and/or DNA
methylation levels could be the signals that trigger the senescence response to replicative aging in human fibroblasts. As is the case with
global DNA methylation levels, DNAMeTase activity was shown to decrease
in an age-dependent manner (21). This observation supports
the idea that this protein is involved in the division counting
mechanism. However, it remains to be determined whether the DNAMeTase
participation in the determination of cellular life span is mediated by
its methyltransferase activity and/or its capacity for
methylation-independent transcriptional regulation.