From the Department of Pharmacology, McGill University, 3655 Drummond Street, Montreal, Quebec H3G 1Y6, Canada
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ABSTRACT |
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The study of the biological role of DNA
methyltransferase (DNA MeTase) has been impeded by the lack of direct
and specific inhibitors. This report describes the design of potent DNA
based antagonists of DNA MeTase and their utilization to define the interactions of DNA MeTase with its substrate and to study its biological role. We demonstrate that the size, secondary structure, hemimethylation, and phosphorothioate modification strongly affect the
antagonists interaction with DNA MeTase whereas base substitutions do
not have a significant effect. To study whether DNA MeTase is critical
for cellular transformation, human lung non-small carcinoma cells were
treated with the DNA MeTase antagonists. Ex vivo, hairpin
inhibitors of DNA MeTase are localized to the cell nucleus in lung
cancer cells. They inhibit DNA MeTase, cell growth, and anchorage
independent growth (an indicator of tumorigenesis in cell culture) in a
dose-dependent manner. The inhibitors developed in this
study are the first documented example of direct inhibitors of DNA
MeTase in living cells and of modified oligonucleotides as bona fide
antagonists of critical cellular proteins.
DNA-binding proteins that regulate gene expression play an
important biological role and are potentially attractive therapeutic targets. However, the study of their role in different physiological and pathological processes has been hindered by the lack of specific inhibitors. These proteins are especially appealing as drug targets because their ligand is a DNA sequence that can be identified by
standard molecular biology techniques and can be synthesized and
modified by well established chemistries (1). In addition, recent
observations suggest that double-stranded DNA-based oligonucleotides bearing transcription factor recognition sites can be delivered into
cells in culture and in vivo and exhibit pharmacological effects (2, 3). A major limitation of this approach is that oligonucleotide antagonists that are identical to the transcription factors cognate site act as stochiometric competitors. Therefore, very
high intracellular concentrations are required to effectively engage
all the transcription factor available in the cell at all times. An
ideal DNA-binding protein antagonist should exhibit higher affinity to
the protein than the cognate sequence and bear a slow off rate. In this
report we have tested the hypothesis that DNA-based inhibitors of
DNA-binding proteins that address these requirements could be
developed, using the DNA methyltransferase enzyme (DNA
MeTase)1 as a model
DNA-binding protein.
Basic oncogenic pathways such as the Ras-Jun signaling pathway have
been shown to up-regulate DNA MeTase mRNA (4-6) and the hyperactivation of DNA MeTase observed in many cancer cells (7, 8)
occurs in parallel with the development of the aberrant patterns of DNA
methylation that these cells exhibit (9, 10). A number of studies
suggest that the hyperactivation of DNA MeTase plays a causal role in
oncogenesis. For example, the intraperitoneal injection of antisense
oligonucleotide to DNA MeTase mRNA into LAF/1 mice bearing tumors
derived from the syngeneic tumor cell line Y1 (11) inhibits tumor
growth; and in vivo reduction of DNA MeTase levels by either
5-azaCdR treatment or by bearing one mutated allele of DNA MeTase
reduces the frequency of appearance of intestinal adenomas in the Min
mouse bearing a mutation in the adenomatosis polyposis coli gene
(12).
Uncovering the biological role of DNA MeTase in vivo
requires the availability of specific DNA MeTase inhibitors especially since mice bearing a null mutation of the DNA MeTase die at
midgestation (13). The most established inhibitors of DNA MeTase are
nucleoside analogs such as 5-fluorouracil and 5-azaCdR (14), which are believed to covalently trap the DNA MeTase after incorporation into DNA
(15). It has been recently shown that this mechanism of action is
responsible for the cytotoxic and mutagenic side effects of 5-azaCdR
(16, 17), which seriously limits their ability to be used in either
therapeutics or research (18). One direct inhibitor of DNA MeTase is
S-adenosylhomocysteine (19); however,
S-adenosylhomocysteine is not a specific inhibitor of DNA
methylation and might inhibit other methylation reactions in the cell.
We reasoned that oligonucleotide-based DNA analogs that bind the DNA
MeTase but are not acceptors of the methyl group will form a high
affinity stable intermediate with DNA MeTase and will be potent
inhibitors of DNA MeTase. In this paper, we describe the design of self
complementary oligonucleotides that form "hairpin" structures and
demonstrate that a combination of modifications of the DNA sequence,
methylation status, and phosphate backbone of these oligonucleotides
results in modified substrates that act as potent and efficacious
direct inhibitors of DNA MeTase.
Oligonucleotide Synthesis--
All oligonucleotides were
synthesized at Hybridon Inc. using standard phosphoramidite chemistry
as described previously, purified on a 20% denaturing PAGE, eluted by
salt extraction, denatured by boiling, and reannealed by slow cooling
to room temperature, and dissolved in water at a final concentration of
1 mM.
Cell Culture--
A549 non-small cell lung carcinoma cells
(ATCC, CCL 185) were grown in Dulbecco's modified Eagle's medium
(with low glucose) supplemented with 10% fetal calf serum and 2 mM glutamine. H446 lung carcinoma cells (ATCC, HTB 171)
were grown in RPMI 1640 medium with 5% fetal calf serum. To treat A549
cells with hairpin oligonucleotides, cells were plated 18 h before
treatment at a concentration of 3 × 105 cells per
10-cm plate. The relevant hairpin oligonucleotides were mixed with 30 µl of Lipofectin (2 mg/ml, Life Technologies, Inc.) and 4 ml of
OPTI-MEM serum-free medium and left at room temperature for 15 min and
then added to cells. The hairpin containing medium was removed from the
cells and replaced with regular growth medium after 4 h. The
treatment was repeated 3 times at 24-h intervals. The cells were split
after the third treatment and equal numbers of cells were plated on
10-cm tissue culture plates and harvested after 24 h.
DNA MeTase Activity Assay--
DNA MeTase activity was assayed
using nuclear extracts (5 µg of total protein) prepared from human
lung carcinoma cell line H446 as described previously (20). The
reaction mixture (final volume of 30 µl) included the indicated
concentrations of oligonucleotides in a buffer containing 10 mM Tris-HCl, 25% glycerol, 5 mM
MgCl2, 0.5 mM phenylmethylsulfonyl fluoride,
0.15 M NaCl, 1 µl of
S-[methyl-3H]adenosyl-L-methionine
(78.9 Ci/mmol, Amersham) (AdoMet) as a methyl donor, and 0.2 µM of a synthetic hemimethylated double-stranded oligonucleotide substrate as a methyl acceptor as described previously (20). Following 2 h incubation at 37 °C the reaction was
inactivated by heating at 65 °C for 5 min and the incorporation of
methyl groups into the DNA substrate was determined by scintillation counting of trichloroacetic acid precipitable counts. For each experiment, the background counts reflecting methylation of other macromolecules present in the nuclear extract as well as endogenous DNA
methylation were determined by incubating the nuclear extract under the
same conditions in the absence of the hemimethylated substrate. In a
typical experiment the background counts obtained with nuclear extract
alone were around 3000 dpm. The background counts were subtracted from
the values obtained per each point. The DNA methyltransferase reaction
is linear for 3 h under these conditions. The results are
presented as an average of three determinations and the apparent
constant of inhibition was determined by curve fitting of the
dose-response curve using Sigma Plot software.
Purification of DNA MeTase from A549 Cells by
Chromatography--
DNA MeTase was purified from A549 human lung
carcinoma cells. Nuclear extracts were prepared from cultures at near
confluence. Nuclei were isolated by resuspending and incubating the
cells in buffer A (100 µl/107 cells: 10 mM
Tris, pH 8.0, 1.5 mM MgCl2, 5 mM
KCl, and 0.5% Nonidet P-40) at 4 °C for 10 min, which was followed
by centrifugation at 1,000 × g for 10 min. The nuclear
pellet were resuspended in buffer A (400 µl) and collected as above.
Nuclear proteins were extracted by resuspending and incubating the
nuclear pellet in buffer B (30 µl/107 cells: 20 mM Tris, pH 8.0, 25% glycerol, 0.2 mM EDTA,
and 0.4 mM NaCl) at 4 °C for 15 min, followed by
centrifugation at 10,000 × g for 30 min to separate
the nuclear extract in the supernatant from the chromatin pellet.
Nuclear extracts were stored at Purification of Recombinant Human DNA MeTase--
The cDNA
bearing the coding sequences of human DNA MeTase was generated as
follows. Subdomains of the human DNA-cytosine-5-methyltransferase cDNA were generated by reverse transcriptase-polymerase chain reaction from 1 µg of total RNA prepared from the human small lung
carcinoma cell line H446 using the following set of primers: a,
5'-ccccatcggtttccgcgcgaaaa-3'(sense; 168-190) and
5'-gcatctgccattcccactct-3' (antisense; 611-592); b,
5'-ttatccgaggagggctacct-3' (sense; 454-473) and
5'-cccttccctttgtttccagggc-3' (antisense; 3573-3551); c,
5'-ggcaagggaaaagggaaggg-3' (sense; 3571-3590) and
5'-gtccttagcagcttcctcct-3' (antisense; 5085-5066). The nucleotide
positions are according to accession number X63692. The polymerase
chain reaction products were cloned in pCR3.1 vector (InVitrogen) and
the sequence of the cDNAs was verified by dideoxy chain termination
method using a T7 DNA sequencing kit (Pharmacia) and alignment to the
published human DNA MeTase sequence (21). To generate the DNA MeTase
construct we first cleaved the pCR 3.1 bearing fragment c with
EcoRI, the fragment was blunted and ligated to a pCR3.1
vector bearing fragment b which was cleaved at the 3' EcoRV
site. This construct was then cleaved with XbaI and ligated
it into a pCR3.1 vector bearing fragment a which was cleaved at an
internal XbaI site to form the reassembled MeTase construct.
This plasmid can direct expression from the bacteriophage T7 or the
mammalian cytomegalovirus promoter. Escherichia coli BL21 DE
3 cells (expressing T7 polymerase under a Lac promoter) were
transformed with plasmid pCR 3.1MeTase and grown in 2 liters of Luria
broth medium at 37 °C up to an OD600 of 0.6. The cells
were induced with 1 mM
isopropyl-1-thio- Electrophoretic Mobility Shift Assay--
To identify
DNA-protein complexes formed between DNA MeTase and the hairpin
inhibitors, an electrophoretic mobility shift assay was performed. The
different oligonucleotides were labeled at their 5' with
32P by incubating 5 µM purified
oligonucleotide with 50 µCi of [
The DNA binding reactions contained 20 mM HEPES (pH 7),
12% glycerol, 1 mM EDTA, 4 mM dithothreitol,
0.1% Nonidet P-40, 3 mM MgCl2, 1 µg of
purified human MeTase, 5 µg of bovine serum albumin, 300 µM scrambled phosphorothioate oligonucleotide as a
nonspecific competitor (5'-CGATTCAATCCTCACCTCTC), and the indicated concentration of hairpin oligonucleotide in a final reaction volume of
10 µl. The mixtures were incubated for 30 min at room temperature, and the complexes were resolved by electrophoresis in a 4%
nondenaturing polyacrylamide gel at 4 °C for approximately 3 h
at 220 volts. For competition experiments, a 100-fold molar excess of
the competing oligonucleotide was added to the reaction mixture prior
to adding the labeled test hairpin inhibitor.
For determination of apparent dissociation constants
(Kd), the level of complex formation at different
substrate concentrations was quantified with a PhosphorImager and
plotted against the substrate concentration. The Kd
was calculated using the Sigma plot software. To determine the
stability of the complex formed with the hemimethylated hairpin
(koff), a binding reaction mixture of 100 µl
was prepared with the same final concentrations described above. After
an initial incubation time of 30 min, 100-fold excess of competitor was
added (time 0) at room temperature and 10 µl of the binding reaction
were removed at each time point and loaded on a 5% nondenaturing
polyacrylamide gel.
Coupled Transcription Translation of Human DNA MeTase--
Human
DNA MeTase was synthesized by coupled transcription-translation, with
the Promega TNT reticulocyte lysate kit, from 2 µg of human DNA
MeTase cDNA cloned into pCR 3.1 using T7 RNA polymerase and 20 µCi of [35S]methionine (1,000 Ci/mmol) in a 50-µl
reaction volume in accordance with the manufacturer's instructions.
Preparation of Biotinylated Hemimethylated Hairpin Coupled to
Streptavidin-coated Magnetic Beads and Binding DNA MeTase to Magnetic
Beads Bound Hairpin--
Streptavidin-coated magnetic beads (Dynabeads
M-280 streptavidin) were purchased from Dynal. The hemimethylated
5'-biotin phosphorothioate hairpin oligonucleotide had an identical
sequence to oligo 3018 and was synthesized using phosphoramidite
chemistry on a Biosearch model 8700 automated synthesizer and was
purified by high performance liquid chromatography using a
phenyl-Sepharose column followed by DEAE 5PW anion exchange
chromatography. The oligonucleotide (at a final concentration of 3 µM) was incubated with 5 mg of beads in a buffer
containing 10 mM Tris-HCl EDTA (pH 7.4) and 1 M
NaCl for 10 min at room temperature. The beads were then washed and
resuspended in 500 µl of the same buffer. The final concentration of
the oligo bound to the beads was 150 pmol/mg and was calculated by
measuring the optical density at 260 nm of the supernatant before and
after binding of beads.
To assay DNA MeTase binding to the hairpin bound beads, 50 µg of
nuclear extract or 3 µl of in vitro translated DNA MeTase were preincubated in a 30-µl reaction mixture including nonspecific or specific competitor oligonucleotide (100 µM), 10 mM Tris-HCl EDTA (pH 7.4), protease inhibitors
(phenylmethylsulfonyl fluoride, aprotinin, sodium vanadate, 10 µg),
and 40 mM NaCl. At this point the mixture was applied onto
500 µg of hairpin bound beads for 5 min. The bound fraction and the
unbound supernatant were separated by a magnet and the bound beads were
washed twice with 100 µl of 10 mM Tris-HCl EDTA (pH 7.4)
buffer followed by two washes in a 1 M NaCl containing
buffer. After washing, the bead bound fraction was resuspended in 30 µl of 10 mM Tris-HCl EDTA (pH 7.4), 0.1% SDS solution
and boiled for 5 min. The different fractions were loaded onto an
SDS-PAGE gel, dried, and exposed to autoradiography.
Assay of DNA Strand Extension Activity in Nuclear
Extracts--
SK-Bluescript plasmid (50 ng, Stratagene) was denatured
by boiling for 10 min in presence of a hexanucleotide mixture
(Boehringer-Mannheim) in the presence of Boehringer primer extension
buffer, and was then annealed with the random primers by slow cooling
to room temperature. 0.25 µM dNTPs ( Inhibition of HpaII MeTase Activity--
SK-Bluescript plasmid
(1 µg, Stratagene) was incubated for 1 h at 37 °C with 10 units of HpaII MeTase (New England Biolabs) and 80 mM S-adenosylmethionine, in the buffer
recommended by the manufacturer, in the presence of the indicated
concentrations of hairpin or scrambled oligonucleotide. The reaction
was terminated and the enzyme was heat inactivated at 65 °C for 20 min. The methylated plasmid was subjected to digestion with 10 units of
HpaII restriction enzyme in 40 µl of the appropriate
buffer for 30 min at 37 °C and the digestion products were analyzed
on a 0.8% agarose gel which was subjected to Southern blotting and
hybridization with a 32P-labeled SK probe.
Bisulfite Mapping--
Bisulfite mapping was performed as
described previously (22). The primers used for the Human
MyoD first exon (GenBank accession X56677) were: MyoD IN1'
5'- GTTTTTTTTGTTTTTTTGTTATAA-3' (position 173-195), MyoD OUT1'
5'-TTGTTAGTATTTTGTTATTTATAG-3' (position 8-21), MyoD IN2'
5'-CTCAAAAACCTCATTTACTTT-3' (position 506-483), MyoD OUT2'
5'-CAAAATCTCCACCTTAAACA-3' (position 569-549).
Design Principles--
Our general goal was to design a modified
DNA MeTase substrate that will bind the DNA MeTase with higher affinity
than the unmodified sequence, will not serve as a methyl acceptor in
the reaction, but will occupy the enzyme. The following principles were
used in our initial design of modified substrates. First, the
preferable substrate for vertebrate DNA MeTase is a hemimethylated CpG
dinucleotide contained in double-stranded DNA (23). However, it stands
to reason that cointroduction in vivo of two annealed oligonucleotides without disturbing their interaction is an arduous task. There is documented evidence, however, that single-stranded oligonucleotides could be delivered ex vivo and in
vivo (2). We therefore opted at designing self-complementary
single-stranded oligonucleotides that can form hairpin structures and
enable the formation of a hemimethylated duplex (see Table
II for prototype structures). A
hemimethylated DNA duplex forms, however, only a low affinity complex
with mammalian DNA MeTase (24, 25). It is therefore clear that certain
modifications will be required to generate a potent inhibitor of DNA
MeTase. Altering the recognition sequence in the hemimethylated CpG
dinucleotide pair can significantly alter the affinity of the enzyme to
the substrate and the rate of methylation (26). For example, it has
been shown that structures associated with more weakly stacked cytosine
rings, such as mispaired or abasic duplexes and other unusual DNA
structures, are preferentially methylated by the human DNA MeTase (26,
27).
We designed our hairpins to recapitulate the natural substrate. The 5'
arm of the hairpin bearing methylated CpGs mimics the parental
methylation guiding strand whereas the 3' arm of the hairpin mimics the
nascent methyl-acceptor strand of replicating DNA (Table II). As our
goal was to design an inhibitor of DNA MeTase, we tested the hypothesis
that introducing changes similar to those previously described (26, 27)
to the methyl acceptor strand will increase the time of occupancy of
the enzyme by the substrate as well as inhibit methyl transfer. In
addition, very little is known about the interactions of the DNA MeTase
and the phosphate and sugar components of the backbone. We reasoned
that these interactions might be critical in determining the ability of
the enzyme to interact with the substrate as well as its ability to
catalyze the methyl transfer.
Modifications of the Phosphate Backbone and the State of
Methylation Strongly Affect the Potency of Hairpin DNA MeTase
Inhibitors--
Based on these principles, a set of single-stranded
oligonucleotides to act as substrates for DNA MeTase were synthesized (Tables II and III). Both hemimethylated
and nonmethylated variants were synthesized as well as various
substitutions to the C located opposite the methylated CpG
dinucleotide. The two CpG sites in each oligonucleotide were separated
by 4 bases to avoid tandem CpGs which were previously shown to be poor
substrates of DNA MeTase (28). All compounds are expected to form a
hairpin structure based on previously documented published data and
theoretical considerations (29-31). We have verified that these
oligonucleotides form double-stranded structures at 37 °C (data not
shown). The potency of the oligonucleotides as inhibitors of DNA MeTase
was determined by measuring the rate of DNA MeTase-catalyzed transfer of a tritiated methyl group from S-adenosylmethionine to a
standard hemimethylated double-stranded oligonucleotide substrate (20) in a nuclear extract prepared from a human small cell lung carcinoma line H446 in the presence of increasing concentrations of these oligonucleotides. Typical curves are presented in Fig.
1. The ability of the inhibitors to serve
as acceptors of DNA MeTase-catalyzed transfer of methyl groups in the
absence of the standard substrate was determined using saturating
concentrations of the inhibitors (1 µM). Nuclear extracts
were used rather than purified enzyme to recapitulate the situation
in vivo where DNA MeTase functions in the presence of other
nuclear proteins and to ascertain that the inhibitors are effective in
the nuclear milieu.
The following conclusions, regarding the structure function
relationship of DNA MeTase antagonists, were derived from the screening
of the different DNA MeTase substrates shown in Table II. First, a
non-modified hemimethylated hairpin oligonucleotide (3048) is a poor
inhibitor of DNA MeTase demonstrating, as predicted in the
introduction, that competitive inhibition using a cognate site of a
DNA-binding protein is inadequate. Second, modification of the
recognition sequence of DNA MeTase, by altering one methyl acceptor CpG
site to inosine (IpG) and removing its methyl acceptor capacity, does
not increase the potency of the inhibitor (3046). Third, modifying the
hairpin phosphate backbone by replacing one of the oxygen groups with a
thiol group (3016, 3018) abolishes its methyl acceptor capacity and
results in potent inhibition (Ki values at the
30-65 nM range). Fourth, hemimethylation increases the
potency of hairpin inhibitors (Table III and Fig. 1; 3016, 3018 versus 3056, 3093). Hemimethylation of a single 5' site
resulted in intermediate potency of the inhibitor (3050 versus 3018 and 3056), suggesting that methylation of the
second site in the upper arm of the hairpin affects the affinity of the MeTase to the substrate. Fifth, a cognate methyl CpG dinucleotide is
required opposite the methyl acceptor site (Table III, 3044 versus 3017) whereas thymidine is adequate in the second
3'-methyl guiding site (Table III, 3019 versus 3016) as has
previously been suggested (26). Sixth, surprisingly, a fully methylated
phosphorothioate hairpin (3191) is a potent inhibitor of DNA MeTase,
suggesting high affinity recognition of a CpG site which is methylated
on both strands by the DNA MeTase (Table III). Seventh, methylation at
cytosines found in CpC dinucleotides located upstream to the CpG
dinucleotide (3092) enhances 4-fold the potency of the inhibitor relative to the unmethylated counterpart (3093) suggesting that the DNA
MeTase can recognize noncanonical methylated cytosines. Eighth,
backbone modifications affect the interaction of DNA MeTase with the
methyl acceptor site since phosphorothioate modification of 9 bases in
the 3'-methyl acceptor arm of a hemimethylated hairpin (3062) is
sufficient to confer high affinity antagonism of DNA MeTase whereas
phosphorothioate modification of the methyl acceptor arm that does not
include the first methyl acceptor site (3061 and 3063) is inadequate.
Ninth, modification of the sugar moiety by 2' O-methylation
(3060 versus 3016) abolishes both the DNA MeTase inhibitory
as well as methyl-acceptor activity suggesting an interaction between
the DNA MeTase and the deoxyribose component of DNA. Alternatively, it
is possible that the ribose-O-methyl groups indirectly
affect MeTase-oligo interaction. Tenth, substituting the methyl
acceptor residue to either uracil (3017), or inosine (3018 and 3062),
or a fluoro substitution (3005), does not significantly reduce or
increase its potency as an inhibitor. However, an abasic site (3053)
reduces the potency of inhibition (Ki of 350 nM). In summary, this structure-activity relationship study suggests that the interaction of DNA MeTase with the methyl acceptor site is influenced by the phosphate backbone, the presence of a
hairpin, and its methylation state but not by the identity of the
acceptor base. This study has identified potent inhibitors of DNA
MeTase as well as nonactive analogs.
DNA MeTase Hairpin Antagonists Specifically Inhibit DNA MeTase but
Not Other Proteins Interacting with DNA--
To determine whether the
active hemimethylated hairpin inhibits specifically mammalian DNA
MeTase but not other DNA interacting proteins, we determined whether
increasing concentrations of the active hairpin (3018) or the inactive
hairpin (3060) will inhibit extension of primed DNA by DNA
polymerase(s) in human nuclear extracts. As observed in Fig.
2A, there is only a small
inhibition of DNA extension activity by the active 3018 hairpin in
comparison with the inactive hairpin.
The hairpins 3017, 3018, and 3044 do not specifically inhibit
HpaII MeTase as measured by the ability of HpaII
MeTase to confer resistance to HpaII cleavage. No inhibition
of HpaII MeTase by hairpins 3017, 3018, and 3044 are
observed up to 100 nM, a concentration at which 80-90% of
mammalian DNA MeTase is inhibited (Fig. 2B). We redetermined the
Ki value of 3016 using purified mammalian DNA MeTase
to demonstrate that the inability of the hairpins to inhibit
HpaII MeTase was not due to the absence of some component found in the nuclear extracts present when the methylase assays shown
in Fig. 1 were performed resulting in the calculated
Ki shown in Table II. The Ki
value obtained was similar (data not shown) indicating that 3016 interacts specifically with human DNA MeTase, but not with
HpaII DNA MeTase. Similarly, these hairpins do not inhibit
EcoRI MeTase (data not shown). The data presented here is
consistent with the hypothesis that oligonucleotide 3018 specifically
inhibits mammalian DNA MeTase but not other CpG MeTases such as
HpaII, other non-CpG MeTases such as EcoRI, or
other DNA modifying proteins such as DNA polymerase.
Hairpin Inhibitors of DNA MeTase Form a Stable Complex with the
Enzyme--
One possible mechanism explaining the inhibitory effect of
the hairpin oligonucleotides described above is that they stabilize a
transition state complex with the enzyme. Since they are not acceptors
of a methyl group as shown above, the enzyme is not removed from the
substrate as it normally is following a methyl transfer, and instead
remains stably bound to it. To test this hypothesis, we incubated a
nuclear extract prepared from human non-small cell lung carcinoma H446
with a hemimethylated hairpin (3018) that had a biotin moiety at its 5'
end. The oligonucleotide-bound proteins were separated from the unbound
supernatant using avidin-coated magnetic beads. Following a wash with a
high salt buffer, the hairpin bound fraction was eluted from the
hairpin bound beads by boiling in an 0.1% SDS buffer. The different
fractions were subjected to a Western blot analysis and the membrane
was reacted with an antibody directed against the catalytic domain of
DNA MeTase (Fig. 3A). The
results demonstrate that DNA MeTase is bound to the hairpin as
indicated by its absence in the supernatant fraction, that it is not
eluted by high salt (1 M NaCl) and that it is only eluted
from the hairpin by boiling (B). This data is consistent
with the hypothesis that DNA MeTase in nuclear extracts forms a stable
noncovalent complex with a hemimethylated hairpin oligonucleotide. To
verify that other nuclear proteins do not form similar stable complexes
with DNA MeTase, we reacted the membrane against a retinoblastoma
protein (Rb) specific antibody. As observed in Fig. 3A, the
Rb protein is fully washed away (W) and none is stably bound
to the hairpin as evidenced by its absence in the boiled fraction
(B).
To further ascertain that DNA MeTase stably binds hemimethylated
hairpins, we in vitro transcribed and translated human DNA MeTase in the presence of [35S]methionine and reacted it
with the biotinylated hairpin as described above. As seen in Fig.
3A (TNT), the in vitro translated DNA
MeTase is stably bound to the hairpin as indicated by its elution in the boiled fraction (B). To exclude the possibility that the
binding of the in vitro translated DNA MeTase reflects some
unknown interactions with proteins present in the in vitro
translation reaction mixture, we expressed recombinant DNA MeTase in
E. coli, purified it to homogeneity by chromatography as
described under "Experimental Procedures," bound it to
32P-labeled hemimethylated oligo 3016 and resolved the
complex formed by nondenaturing acrylamide gel electrophoresis. As
indicated in Fig. 3B, both in vitro translated
DNA MeTase and purified recombinant DNA MeTase form identical
DNA-protein complexes.
AdoMet Is Not Required for Generating a Stable Complex of DNA
MeTase with the Substrate--
Previous studies using
fluoro-substituted oligonucleotides have shown that covalent binding of
DNA MeTase to the substrate occurs only in the presence of the methyl
donor AdoMet (24). Our binding assays are performed in absence of
AdoMet. As demonstrated in Fig. 3C, a DNA MeTase complex is
formed whether or not AdoMet is present in the binding reaction
mixture. This observation is consistent with the hypothesis suggested
above that DNA MeTase forms a stable but not a covalent complex with
the hairpin oligonucleotides. Alternatively, it is also possible that
the direct inhibitor forms a stable complex with the product of the
methylation reaction resulting in the absence of turnover which would
also appear as low enzyme activity when AdoMet is present.
Specificity of DNA MeTase Hairpin Interaction--
To determine
whether the stable complexes formed between DNA MeTase and the
hemimethylated hairpin are specific, we determined whether these
complexes could be competed out by a cold excess of either specific or
nonspecific oligonucleotides. First, we preincubated an A549 nuclear
extract with a 40-fold excess (100 µM) of a nonspecific
phosphorothioate single-stranded oligonucleotide (Scr), the
hemimethylated hairpin oligonucleotide 3018, or with no competitor. A
biotinylated hemimethylated hairpin bound to avidin-coated magnetic
beads was then added to the reaction mixture and the different
fractions of bound and unbound proteins were separated as described
above. As a control, avidin-coated magnetic beads that were not bound
to the hemimethylated hairpin were also tested (beads only). The
hairpin-bound and unbound fractions were subjected to a Western blot
analysis and DNA MeTase was visualized with a DNA MeTase antibody. The
results of such an experiment presented in Fig.
4A show that binding of DNA
MeTase to the hemimethylated hairpin is not competed out by
preincubation with a nonspecific phosphorothioate oligonucleotide as
indicated by the fact that DNA MeTase is eluted only in the boiled
fraction. However, the specific hemimethylated hairpin oligonucleotide
(3018) efficiently competes out the binding of DNA MeTase to the
biotinylated hemimethylated hairpin as evidenced by its elution in the
supernatant fraction. These results support the hypothesis that the
interaction of DNA MeTase and the hemimethylated hairpin is
specific.
We extended our study of the specificity of DNA MeTase and hairpin
interactions by determining the ability of the different classes of
hairpins studied in Tables II and III to compete out the binding of
biotinylated hemimethylated hairpin to in vitro translated
human DNA MeTase. As observed in Fig. 4B,
phosphorothioate-modified hemimethylated hairpins bearing either
inosine (3018) or cytosine (3016) in the methyl acceptor site 1 effectively compete out the binding of DNA MeTase as evidenced by the
presence of DNA MeTase exclusively in the supernatant fraction. The 2'
O-methylated and phosphorothioate-modified hemimethylated
hairpin (3060), which does not inhibit DNA MeTase, does not bind the
DNA MeTase, as evidenced by the presence of DNA MeTase exclusively in
the bound fraction. Similarly, the nonphosphorothioate hemimethylated
hairpin 3046, which is not an inhibitor of DNA MeTase (Table II), does not bind in vitro translated DNA MeTase. The nonmethylated
hairpin 3093 which inhibits DNA MeTase at lower potency (Table III) is a partial competitor as indicated by the presence of DNA MeTase in both
the bound and the supernatant fractions. The nonmethylated inosine-modified hairpin 3056 which does not inhibit DNA MeTase at the
concentrations studied in Table III is a weaker competitor as evidenced
by the large fraction of DNA MeTase that remains bound to the
hemimethylated hairpin after preincubation with this oligonucleotide.
These results were further confirmed by determining the ability of the
32P-labeled hairpins to form a DNA-protein complex with
purified human DNA MeTase as determined by an electrophoretic mobility shift assay (Fig. 4C). This assay measures DNA-protein
interaction per se but not the stable complex formation that
is disrupted only by boiling, which is measured by the assay described
above. As evidenced in Fig. 3C, oligonucleotides that
inhibit DNA MeTase (Table II) form a DNA-protein complex (3016, 3018).
Oligonucleotides that do not inhibit DNA MeTase (Table II and III) do
not form a DNA-protein complex (3061, 3048, 3006, 3060, and Scr).
However, nonmethylated hairpin 3093 which is not a potent inhibitor of DNA MeTase (Table III) can still bind it (Fig. 4, C and
D). Binding of the nonmethylated hairpin competes out the
binding of DNA MeTase to both nonmethylated and methylated hairpin.
Oligonucleotides that do not inhibit DNA MeTase (3048, 3060, 3061, and
Scr) do not compete out the binding of either methylated or
nonmethylated hairpins.
This data is consistent with the ability of mammalian DNA MeTase to
bind and methylate nonmethylated DNA (32). Whereas hemimethylation increases the rate of methylation (32) and the ability of modified DNA
hairpins to inhibit DNA MeTase (table III), the ability to discriminate
de novo and maintenance activities cannot be explained just
by the differential capacity to bind the substrate. Both, methylated
and nonmethylated hairpins form similar specific and identical
DNA-protein complexes with DNA MeTase as demonstrated by the
competition assay shown in Fig. 4D. However, a difference is
observed in the ability of DNA MeTase to form stable complexes with
methylated and nonmethylated hairpins (which are disrupted only by
boiling) as determined by the biotinylated hairpin assay described
above (Fig. 4B, 3056, 3093 versus 3018 and 3016).
Correlation of the Potency of Binding DNA MeTase, Inhibition of
Enzymatic Activity, and Forming of a Stable DNA MeTase Complex by
Hairpin Inhibitors--
To test the hypothesis that the potency of
inhibition of DNA MeTase by hemimethylated hairpins is dependent on its
binding affinity to DNA MeTase, we compared the apparent
Kd of hairpin 3016 for binding purified DNA MeTase
as determined by a electrophoretic mobility shift assay (Fig.
5A, graphed in Fig. 6) and the apparent Ki
for inhibiting DNA MeTase activity (Figs. 6 and 1). Both assays showed
similar dose dependence (Fig. 6). The Kd for binding
and Ki for inhibition of enzymatic activity are
around 60-80 nM (Fig. 6). To further test the hypothesis
that the hemimethylated hairpin forms a stable complex with DNA MeTase,
we first bound purified recombinant DNA MeTase with
32P-labeled hemimethylated hairpin (3018) and then
challenged it with a thousand fold excess of cold hemimethylated
hairpin. As observed in Fig. 5B, the 32P-labeled
hemimethylated hairpin oligonucleotide remains bound to the DNA MeTase
up to 90 min after challenging with cold oligonucleotides. This is
consistent with a slow koff of the
hemimethylated hairpin for DNA MeTase.
In summary, the mechanism of action of the phosphorothioate-modified
hemimethylated hairpin is consistent with the guiding principles
underlying our design of DNA MeTase inhibitors; a potent DNA MeTase
inhibitor exhibits high affinity binding to the enzyme and forms a
stable intermediate DNA MeTase complex but is at best a poor acceptor
of a methyl group.
Ex Vivo Activity of Hairpin Inhibitors of DNA MeTase: Inhibition of
DNA MeTase and Tumorigenesis--
It has been suggested that DNA
MeTase inhibitors might be used therapeutically as antitumor agents
(33, 34). We have previously shown that inhibition of DNA MeTase by
antisense oligonucleotides in living cells or in tumors in
vivo inhibits tumor growth (11). A critical question is whether
the hairpin inhibitors developed in this study can be delivered into
the nucleus, the subcellular site of DNA MeTase, whether they can
stably bind and inhibit DNA MeTase in living cells and whether they can
inhibit the growth of cancer cell lines?
Following a dose-response analysis of inhibition of DNA MeTase by fully
methylated, hemimethylated, nonmethylated, and
2'-O-methyl-modified hairpin analogs as shown in Fig. 1, we
have selected an active hemimethylated phosphorothioate hairpin (3016, Ki = 65 nM) and an inactive
hemimethylated phosphorothioate 2'-O-methyl-modified hairpin
analog (3060, Ki >1 µM) for ex
vivo studies. Both, the control and active hairpins are
hemimethylated and phosphorothioate modified but differ in a
modification of the sugar moiety. Non-small cell lung carcinoma A549
cells were treated with the active and control hairpins at
concentrations of 10-100 nM in the presence of the lipid
carrier, Lipofectin (Life Technologies, Inc.) (Fig. 7). To determine whether the active and
inactive hairpins are delivered into the nucleus, the respective
hairpins were tagged with fluorescin and the localization of the
labeled oligonucleotide was followed by live cell fluorescence
microscopy. As shown in Fig. 7C both the control and the
active hairpins are localized to the nucleus as early as 1 h after
treatment and remain in the nucleus up to 72 h post-transfection
(data not shown).
Previous studies have shown that expression of an antisense mRNA to
DNA MeTase or DNA MeTase antisense oligonucleotides inhibit the growth
of murine adrenocortical carcinoma cells in vitro (11, 35).
We determined whether the direct inhibitors of DNA MeTase developed in
this study might have a similar effect on cell growth. An equal number
of A549 cells were plated and treated with increasing concentrations of
the DNA MeTase inhibitor (3016) and inactive hairpin control (3060).
The cells were photographed (Fig. 7A) and counted (Fig.
7B) 72 h after treatment. As observed in Fig. 7
(A and B), direct inhibitors of DNA MeTase slow
the growth of A549 tumor cells ex vivo, but do not have a
nonspecific toxic effect on the cells. The number of cells increases
after 3 days even in the presence of high dose (100 nM) of
the hairpin inhibitor (3016) but to a lesser extent than nontreated
cells or cells treated with a control oligonucleotide (3060). No
toxicity is observed following 3 days of treatment with direct
inhibitors as judged by morphological examination (Fig. 7A)
or trypan dye exclusion (100% of the cells are viable).
To determine whether the hairpin inhibitor stably inactivated DNA
MeTase in the living cell we assayed the activity of DNA MeTase in
nuclear extracts prepared from hairpin inhibitor (3016) and hairpin
control (3060) treated cells 72 h post-treatment. This assay
detects differences in DNA MeTase activity only if the inhibitor
remains stably bound to the enzyme during the purification protocol. As
we have shown in Fig. 3A that the active hairpin is not
removed from DNA MeTase even by 1 M NaCl, we reasoned that if the inhibitor interacted with the enzyme in the cell by a similar mechanism, it should remain bound to it even after salt extraction of
nuclear extracts. As observed in Fig.
8A, there is a
dose-dependent inhibition in DNA MeTase activity in nuclear
extract prepared from active hairpin (3016)-treated cells but not in
nuclear extracts prepared from control hairpin (3060)-treated
cells.
To determine whether the reduction of DNA MeTase activity affected the
tumorigenic potential of hairpin-treated cells we plated an equal
number of active hairpin and control hairpin-treated cells in soft agar
and onto regular tissue culture plates in parallel. Moreover, the
treated cells could form colonies when plated on regular plastic dishes
suggesting that the inhibitors are not generally toxic. To ensure that
the reduction of soft agar colonies reflects a reversal of
transformation rather than a toxic effect, the number of colonies on
soft agar was divided by the number of colonies formed under regular
tissue culture conditions. As observed in Fig. 8B, treatment
with direct inhibitors of DNA MeTase results in a
dose-dependent inhibition of anchorage independent growth
that roughly parallels the effect observed on DNA MeTase activity shown
in Fig. 8A.
A Western blot and quantification of the signal established that cells
treated with 60 nM of the active hairpin bear a similar level of DNA MeTase protein to the nontreated control (Fig.
9B) which is consistent with
the hypothesis that the reduction in MeTase activity observed in
extracts prepared from hairpin-treated cells is a consequence of stable
binding of the inhibitor to the enzyme.
Common wisdom suggests that inhibitors of DNA MeTase should cause
demethylation of genomic DNA. In contrast, a comparison of the genomic
level of methylation of CpG dinucleotides in cells treated with oligo
3016 and the control oligo 3060 using a nearest neighbor analysis has
revealed no significant differences (data not shown). However, the
results demonstrated in Fig. 9A show a limited demethylation
of two HpaII sites located in the third exon of the
retinoblastoma gene. As indicated in Fig. 9A, an expected 1-kikobase HpaII fragment appears after treating the cells
with 10 nM hairpin 3016 and a second ~0.5-kilobase
fragment is observed in cells treated with 60 nM of the
active hairpin 3016 but not in cells treated with the control hairpin 3060.
To further test the hypothesis that DNA MeTase inhibitor treatment
results in alterations in the DNA methylation pattern, we analyzed the
state of methylation of a CG-rich exon of MyoD using bisulfite mapping.
A summary of such an analysis presented in Fig. 9C shows
that out of 41 CG sites in the region of MyoD analyzed 11 sites are
less methylated, 10 sites are more methylated, while the remainder are
unchanged. These results demonstrate that a reduction of DNA MeTase
activity in the nuclei by direct inhibitors of DNA MeTase does not
result in a broad inhibition of DNA methylation. Instead, the local
changes in methylation at the specific sites observed indicate that the
DNA methylation pattern is destabilized. We have similarly observed
that in murine cells treated with a DNA MeTase antisense
oligonucleotide hypomethylation of some sites was accompanied by
hypermethylation of other
sites.2
A large number of DNA-binding proteins have been shown to play
important physiological and pathophysiological roles. What makes these
proteins excellent drug target candidates is the fact that their ligand
is a specific DNA sequence. Therefore, a short double-stranded
oligonucleotide bearing the protein recognition sequence could compete
with the cognate sequence in the genome for binding to the protein and
serve as a bona fide antagonist.
In addition to their specificity, the utilization of short DNA
sequences as antagonists is attractive because of the relative ease by
which these oligonucleotides can now be synthesized (1). Moreover, an
already growing body of in vivo and clinical data provides
an extensive base of information on the pharmacokinetics of
oligonucleotides as well as their general nonspecific toxicity. However, a potential disadvantage of the use of competitive inhibitors of DNA-binding proteins, especially in a clinical situation, is the
requirement for the accumulation of large quantities of the oligonucleotide inhibitor in the target cell (3, 36).
In this article we have demonstrated that this drawback can be
mitigated by considering the recognition sequences of DNA-binding proteins as ligands of receptors. The classical pharmacological approach to the design of potent and efficacious antagonists of receptors has been to introduce limited chemical modifications to the
basic ligand structure. The large number of modifications that can be
added to the base sequence or phosphate-sugar backbone of
oligonucleotides allows this same concept to be applied to the design
of DNA-binding protein antagonists.
Using DNA MeTase as a model DNA-binding protein, this study
demonstrates that like traditional receptor antagonists, the
modification of oligonucleotides can significantly increase their
specificity and antagonistic activity. In addition to identifying a
number of lead DNA MeTase antagonists (EC50 of 30-60
nM) we would like to suggest that in the process of
determining their potency of inhibition and the strength of their
interaction with DNA MeTase we have been able to identify three
distinct stages in the interaction of DNA MeTase and its substrate. The
first essential stage involves the binding of DNA MeTase to its
substrate, which does not significantly differentiate between
nonmethylated and hemimethylated substrates. The next stage involves
formation of a stable complex, which does not require AdoMet (Fig.
3C), but discriminates between hemimethylated and
nonmethylated substrates as well as the base composition of the methyl
acceptor site (Fig. 4B). This is followed by a covalent complex, which requires the presence of AdoMet resulting in methyl transfer and followed by the release of the DNA enzyme complex (15, 24,
32). Thus a modified oligonucleotide that binds the DNA substrate with
high affinity, but in distinction from the cognate substrate is not an
acceptor of a methyl group, will remain bound to the enzyme and serve
as a potent and efficacious antagonist of DNA MeTase.
DNA MeTase has been recently proposed to be an important therapeutic
target in cancer as well as other conditions (33, 34). This report has
shown that DNA MeTase antagonists can be delivered into cells, inhibit
DNA MeTase activity in human cancer cells, slow cellular growth, and
inhibit anchorage independent growth, which is an indicator of
tumorigenicity. A number of lines of evidence demonstrate that these
effects are due to a specific inhibition of DNA MeTase. First, the
hairpin oligonucleotides are potent inhibitors of DNA MeTase at the
nanomolar range whereas other phosphorothioate oligonucleotides are not
active even at the micromolar range. Second, there is a good
correlation between the potency of the oligonucleotides and their
specific binding to DNA MeTase. Third, the amount of the inhibitor
(3016) required to reduce the amount of DNA MeTase activity that can be
extracted from treated cells is similar to the amount of the inhibitor
used in the in vitro methylation reactions (nanomolar
range). Fourth, the potent inhibitors do not inhibit other activities
such as DNA synthesis in a nuclear extract or HpaII MeTase.
Fifth, there is a similar dose response for inhibition of DNA MeTase,
cell growth, and anchorage independent growth. Sixth, the inactive analog (3060) has no effect on cell growth and anchorage independent growth. Seventh, whereas the inhibitor of DNA MeTase slows cell growth
it has no nonspecific toxic effects. Eighth, whereas the inhibitor is
localized to the nucleus 1 h after transfection, inhibition of DNA
replication is only observed after 24 h, suggesting that the
oligonucleotides do not have a nonspecific immediate effect such as
inhibiting directly DNA polymerases (data not shown).
Whereas the common wisdom suggests that inhibitors of DNA MeTase should
cause demethylation of genomic DNA, the results reported in this
article report very limited demethylation induced by our inhibitors
(Fig. 9C). However, since the hairpins described here do not
actively remove methyl groups from DNA, they are expected to cause
genome wide demethylation only if DNA replication proceeds at a high
rate. As the hairpins described here effectively inhibited cell growth
we did not expect wide demethylation to be induced by these inhibitors.
Instead these results suggest that DNA methylation patterns reflect a
dynamic balance of DNA MeTase activity, properties of the sequence and
protein interactions at specific sites (34). Reduced methylation of
some sites might disturb this balance, resulting in increased affinity
of the DNA MeTase to other sites.
Certainly one of the future utilities of these inhibitors will
be to determine the mechanism by which an inhibition of DNA MeTase slows cell growth. Two possible explanations for the
antitumorigenic effects observed are: an active DNA MeTase is
an essential component of the replication fork or,
alternatively, the specific demethylation of some sites may
trigger the activation of tumor suppressor genes. Regardless,
this is the first documented example of direct inhibitors of
DNA MeTase inhibiting DNA MeTase in living cells and they have helped to define the DNA-binding site of the DNA MeTase.
Furthermore, the effectiveness of the inhibitors described
here validate the use of modified oligonucleotides as bona fide
antagonists of critical DNA-binding proteins.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
80 °C. Freshly prepared nuclear
extract was diluted to a conductivity equivalent to 0.2 M
NaCl and applied onto a DEAE-Sepharose (Pharmacia) column (1.0 × 5 cm), which was pre-equilibrated with buffer P (10 mM
potassium phosphate, pH 7.5, 1 mM sodium EDTA, 14 mM
-mercaptoethanol, 10% glycerol) containing 0.2 M NaCl. The column was operated at a flow rate of 1 ml/min.
After sample loading, the column was washed with 15 ml of the starting
buffer (buffer P + 0.2 M NaCl) and the proteins were eluted
with 5 ml of a linear NaCl gradient (0.2-1.0 M). 0.5-ml
fractions were collected and assayed for DNA MeTase activity as
described above after desalting through a Microcon 10 (Amicon Inc.)
spin column. DNA MeTase eluted between 0.3 and 0.5 M NaCl.
The pooled active fractions were adjusted to 0.2 M NaCl by
dilution and applied onto a Q-Sepharose column (1.0 × 5 cm),
which had been previously equilibrated with buffer P at a flow rate of
1 ml/min. The column was washed and eluted with a 5 ml of a linear NaCl
gradient of 0.2-1.0 M. 0.5-ml fractions were collected and
assayed for DNA MeTase activity after desalting and concentrating (to a
final volume of 0.2 ml) through a Microcon 10 spin column. MeTase
activity eluted between 0.3 and 0.4 M NaCl. The pooled
active fractions were adjusted to 0.2 M NaCl, loaded onto a
2.0 × 2.0-cm DEAE-Sephacel (Pharmacia) column, and eluted with 10 ml of buffer P containing 0.2 M NaCl. The fractions (0.8 ml) were collected and assayed after concentration to about 200 µl,
for MeTase activity. Activity was detected at fraction 4, which is very
near the void volume (Table I). A silver
staining PAGE analysis revealed one distinct band migrating around 200 kDa. DNA MeTase is an unstable protein in our hands which explains in
part the loss of activity through the purification procedure (Table I).
This loss of activity can explain why the fold purification as
determined by enzymatic assays is only 240-fold whereas only a single
distinct band is identified by a silver-stain PAGE analysis. The
identity of the band as DNA MeTase was verified by a Western blot
analysis of the purified fraction with a previously described anti DNA
MeTase polyclonal antibody (11).
Purification of DNA MeTase from human A549 cells
-galactopyranoside for 6 h. Following
centrifugation at 4000 rpm for 10 min, the cells (10 g) were suspended
in 10 ml of Buffer P, sonicated for 5 min with a burst and a gap of
15 s. The supernatant was separated by centrifugation at 4000 rpm
for 20 min and was blended with the protease inhibitors aprotinin,
leupeptin, and Pefabloc SC (Boehringer-Mannheim) (10 µl each of 1 mg/ml stock solution) and loaded onto a 10-ml phosphocellulose column
equilibrated with Buffer P + 0.2 M NaCl. Active fractions
were identified by a DNA MeTase assay as described above and were
pooled and further purified by Q-Sepharose and DEAE-Sephacel
chromatography as described above for A459 human DNA MeTase.
-32P]ATP (3000 Ci/mmol, Amersham) and 10 units of T4 polynucleotide kinase
(Boerhinger-Mannheim) in a final volume of 50 µl at 37 °C for
2 h. The labeled oligonucleotide was then purified on a 6%
denaturing PAGE to remove the T4 polynucleotide kinase and the
unincorporated [
-32P]ATP, eluted from the gel, ethanol
precipitated, and resuspended in double-distilled water. This mixture
was heated at 90 °C for 5 min and the hairpin was annealed by
allowing the mixture to cool slowly to 25 °C.
dCTP), 50 µCi of
[
-32P]dCTP (3000 Ci/mmol, NEN Life Science Products
Inc.), 5 µg of H446 nuclear extract, and the indicated concentration
of hairpin oligonucleotide or scrambled oligonucleotide were added to
the reaction mixture to a final volume of 30 µl and incubated at
37 °C for 45 min. The incorporation of labeled dCTP into extended DNA was determined by scintillation counting of trichloroacetic acid
precipitable counts.
RESULTS
Modifications of the phosphate backbone determine the potency of
hairpin inhibitors of DNA MeTase
The state of methylation of hairpins affects their potency
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Fig. 1.
Dose-response analysis of inhibition of DNA
MeTase activity by methylated, hemimethylated, nonmethylated and
2'-O-methyl-modified hairpins. DNA MeTase
activity in nuclear extracts (5 µg) was determined in the presence of
increasing concentrations of the indicated hairpins (1-1000
nM) hemimethylated 3016, nonmethylated 3093, fully
methylated 3191, and 2'-O-methyl-modified hemimethylated
3060 hairpins, as described under "Experimental Procedures." The
data presented is an average of three determinations ± S.D. The
Kd was calculated using the Sigma plot
software.
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Fig. 2.
Hairpin inhibitors of DNA MeTase do not
inhibit either DNA extension activity in nuclear extracts or
HpaII MeTase. A, DNA polymerase
activity. DNA extension activity was assayed in nuclear extracts
prepared from A549 cells using random primed SK- plasmid as a primed
template, [ -32P]dCTP and the other three dNTPs in the
presence of increasing concentrations (1-1000 nM) of the
indicated oligonucleotides, the active hairpin 3018 or the inactive
hairpin 3060. A nonspecific phosphorothioate oligonucleotide sequence
(Scr) was used as a control for each concentration of
oligonucleotide. The rate of incorporation of labeled dCTP at each dose
was determined, normalized against the rate of incorporation of dCTP
into DNA with the same dose of nonspecific phosphorothioate
oligonucleotide, and presented as % of control. B,
HpaII MeTase activity. SK- plasmid was incubated with
HpaII and the indicated concentrations of active hairpin
(3017, 3018), inactive hairpin (3044), and scrambled oligonucleotides.
The HpaII-methylated DNA was subjected to digestion with
HpaII restriction enzyme and the digestion products were
subjected to Southern blot analysis using 32P-labeled SK as
a probe.
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Fig. 3.
Hairpin inhibitors of DNA MeTase form a
stable complex with the enzyme. A, binding of a
biotinylated hairpin to DNA MeTase in A549 nuclear extracts. Ten µg
of nuclear extract prepared from A549 cells were incubated with a
avidin-coated magnetic beads bound with biotinylated hemimethylated
hairpin (3018), the bound proteins were separated from the supernatant
(S) using a magnet and the bound proteins were eluted by
either 1 M NaCl (W) or boiling in a 0.1% SDS
solution. The different fractions and nuclear extracts (NE)
were analyzed by PAGE, Western blotted, and reacted with either an
anti-DNA MeTase antibody (23) (MeTase) or an anti-Rb
antibody (Rb C-15, Santa Cruz Biotechnology Inc.). The blots were
developed with ECL kit (Amersham). Human DNA MeTase was translated
in vitro in the presence of [35S]methionine
(TNT), bound to the biotinylated hairpin as described under
"Experimental Procedures." The supernatant after magnetic
separation (S) and the fraction removed from the beads by
boiling (B) were separated on PAGE and exposed to
autoradiography. The arrows indicate the position of
molecular weight standards. B, binding of in
vitro translated human DNA MeTase and recombinant human DNA MeTase
purified from bacterial cells. 1 µM
32P-labeled hemimethylated hairpin 3018 was incubated with
human DNA MeTase purified from bacterial cells (recombinant) as
described under "Experimental Procedures." The complexes were
resolved on nondenaturing PAGE and exposed to autoradiography.
C, AdoMet is not required for binding. 1 µM
32P-labeled hemimethylated hairpin 3018 was incubated with
purified human DNA MeTase (100 ng) in the presence (+) or absence ( )
of 100 µM S-adenosylmethionine. The complexes
were resolved as above and the complexes corresponding to DNA MeTase
were visualized by autoradiography.
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Fig. 4.
Specificity of interaction of DNA MeTase with
hairpin inhibitors. A, A549 nuclear extract. Ten µg
of nuclear extract prepared from A549 cells were preincubated with 100 µM nonspecific phosphorothioate-modified oligonucleotides
(Scr), 100 µM of the hemimethylated hairpin
3018, or no competitor (control). Avidin-coated magnetic
beads bound with biotinylated hemimethylated hairpin (3018) were then
added to the mixture. In one control sample (beads only) unbound
avidin-coated magnetic beads were added. The bound proteins were
separated from the supernatant (S) using a magnet and the
bound proteins were eluted by successive 1 M NaCl salt
elutions (1-3), followed by boiling in a 0.1% SDS solution
(B). The different fractions and nuclear extracts
(NE) were analyzed by PAGE, Western blotted, and reacted
with an anti-DNA MeTase antibody (11). B, specificity of the
stable complex formed between in vitro translated human DNA
MeTase and hemimethylated hairpin (3018).
[35S]Methionine-labeled human DNA MeTase was translated
in vitro (TNT) and preincubated with either no
competitor (none), 1 µM nonspecific
phosphorothioate oligonucleotide (Scramble), or with either
of the hairpins as indicated in the figure. Following 5 min of
preincubation at room temperature, avidin-coated magnetic beads bound
with biotinylated hemimethylated hairpin (3018) were added to the
mixture as described under "Experimental Procedures." The
hairpin-bound and unbound (S) fractions were separated. The
supernatant after magnetic separation (S) and the fraction
removed from the beads by boiling (B) were separated on PAGE
and exposed to autoradiography. Oligo 3175 is identical to oligo 3093 shown in Table III. C, hairpins that inhibit DNA MeTase
activity bind DNA MeTase as determined by an electrophoretic mobility
shift assay. 1 µM 32P-labeled hairpins as
indicated in the figure were incubated with purified human DNA MeTase.
The complexes were resolved as above and the complexes corresponding to
DNA MeTase (indicated by an arrow) were visualized by
autoradiography. 3006 is an inactive oligonucleotide which does not
inhibit DNA MeTase of the sequence
5'm-CGAAmCGTTTTCGTTCG-3'. D,
specificity of binding of hemimethylated (3016) and nonmethylated
(3093) hairpins to DNA MeTase as determined by an electrophoretic mobility shift assay. Purified
human DNA MeTase was preincubated for 20 min with either 10 µM nonspecific phosphorothioate oligonucleotide
(Scramble) or with the hairpins as indicated in the figure
(Competitor oligo) at room temperature. 1 µM
either 32P-labeled hemimethylated (3016) or nonmethylated
(3093) hairpins were then added to the reaction mixture, the complexes
were resolved by nondenaturing PAGE complexes corresponding to DNA
MeTase (indicated by an arrow) were visualized by
autoradiography.
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Fig. 5.
Dose response and stability of DNA MeTase
binding by hairpins. A, DNA MeTase-hairpin binding as a
function of hairpin concentration. Purified human DNA DNA MeTase was
incubated with increasing concentrations of either
32P-labeled hemimethylated (3016) or nonmethylated (3093)
hairpins as indicated in the figure. The DNA-protein complexes were
resolved as above and the complexes corresponding to DNA MeTase
(indicated as DNA MeTase) were visualized by autoradiography. (DNA
MeTase is partially unstable under these conditions and some smaller
fragments than the full MeTase are detected.) B, formation
of a stable complex between hemimethylated hairpin and DNA MeTase. 100 nM 32P-labeled hemimethylated hairpin (3016)
were incubated with purified human DNA MeTase. 100 µM
of the unlabeled hairpin (3016) were then added to the
reaction mixtures and 10-µl samples were removed at different time
points after challenging with the cold oligonucleotide. The complexes
were resolved by nondenaturing PAGE and the band corresponding to DNA
MeTase is visualized by autoradiography.
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Fig. 6.
DNA MeTase binding and inhibition of DNA
MeTase activity as a function of the concentration of hemimethylated
hairpin (3016). DNA MeTase activity in H446 nuclear extracts was
determined as described under "Experimental Procedures" using
hemimethylated substrate,
S-[3H]adenosylmethionine as a methyl donor and
increasing concentrations of the hemimethylated hairpin (1-1000
nM) as described under "Experimental Procedures." The
data presented is an average of three determinations ± S.D.
Binding, a DNA binding assay similar to the one shown in
Fig. 4A was exposed to a PhosphorImaging plate (Fuji,
BAS 2000) and was quantified using a BAS-TR2040 PhosphorImager plate.
The data presented is an average of three determinations ± S.D.
The Kd and Ki were calculated
using the Sigma plot software.
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Fig. 7.
Hemimethylated hairpin inhibit cell growth of
human nonsmall lung carcinoma cells A459 in culture. A,
phase contrast microscopy of A459 cells treated with either 100 nM hairpin or control hairpin oligonucleotides. Phase contrast microscopy at × 100 magnification of A459 cells treated with a hairpin inhibitor of DNA
MeTase and a control hairpin for 72 h. An equal number of A459
cells (1 × 105 cells per/cm plate) were treated with
increasing concentrations (100 nM) of either hemimethylated
hairpin 3016 or the 2'-O-methyl-modified hemimethylated 3060 hairpin control using Lipofectin as a lipid carrier three times every
24 h as described under "Experimental Procedures."
B, cell number. A549 cells were treated with increasing
concentrations of either hemimethylated hairpin 3016 or control hairpin
3060 as described above were harvested 72 h post-initiation of
treatment and counted. The first bar indicates the number of
cells at time 0. The following bars represent the number of cells after
3 days of treatment with either oligonucleotide. C, nuclear
localization of hemimethylated and control hairpins. A549 cells were
treated with a 5' fluorescein-tagged hemimethylated hairpin (3118, identical to 3016) or the control hairpin (3188 identical to 3060) and
the oligonucleotide was visualized by a fluorescence microscope 1 h after adding the oligonucleotide. The fluorescence micrographs are
presented at a magnification of × 400. Whereas most of the
fluorescence is localized to the nucleus, some fluorescence is
diffusely present in the cytosol. Nontransfected cells show as expected
no fluorescence (data not shown).
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Fig. 8.
Inhibition of DNA MeTase activity and
anchorage independent growth on soft agar. A, DNA
MeTase activity in nuclear extracts prepared from treated cells.
Nuclear extracts were prepared from A549 cells treated with increasing
concentrations of either hemimethylated hairpin 3016 or control hairpin
3060 as described above. DNA MeTase activity in 3 µg of each extract
was determined as described under "Experimental Procedures" using a
hemimethylated substrate and
S-[3H]adenosylmethionine as a methyl donor as
described under "Experimental Procedures." The results presented
are an average of three determinations ± S.D. B, an
equal number of viable A549 cells (3000) (as determined by trypan blue
dye exclusion) treated with the hemimethylated hairpin 3016 as above
were seeded onto soft agar in triplicate for determination of anchorage
independent growth as described previously (10) and 300 cells were
plated on regular tissue culture plates to determine the ability of the
treated cells to form colonies. The number of colonies on soft agar was
counted after 3 weeks and normalized to the number of colonies formed
under regular conditions.
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Fig. 9.
Effects of hairpin inhibitors on DNA
methylation. A, analysis of methylation state of
HpaII sites located in the Rb locus. DNA (10 µg) isolated
from A549 cells treated with either increasing concentrations of
hemimethylated hairpin (3016) or control hairpin (3060) were subjected
to HindIII digestion followed by either HpaII
(H) (which cleaves the sequence CCGG when the internal C is
not methylated) or MspI (M) which cleaves the
sequence CCGG even when the internal C is methylated, digestion, 1.5%
agarose gel fractionation, Southern blotting onto a Hybond N+ membrane,
and hybridization with a 32P-labeled 1.4-kilobase
HindIII fragment from plasmid pH3-8 (ATCC 57450). The
position of the probe relative to the physical map of the Rb gene is
underlined. The positions of the HindII fragment
and the expected MspI/HpaII fragments are
indicated. B, Western blot analysis of DNA MeTase protein
extracted from hairpin-treated cells. 50 µg of nuclear extracts
prepared from A459 cells treated with Lipofectin alone, 60 nM hemimethylated hairpin 3016, or control hairpin 3060 were subjected to a Western blot analysis. DNA MeTase is visualized
using a 1:2000 dilution of anti-DNA MeTase antibody (24) and an
enhanced chemiluminescence detection kit (Amersham). Two bands are
visualized. The signal obtained for the hemimethylated treated hairpin
was similar to the signal obtained for Lipofectin treated controls. The
amount of signal corresponding to DNA MeTase (OD arbitrary units) was
normalized to the level of total protein transferred onto the membrane
as determined by Amido Black staining and quantified by scanning (OD
arbitrary units). The values obtained (OD of DNA MeTase signal divided
by OD of the total protein staining): upper band: Lipofectin
alone, 0.81; control hairpin 3060, 0.82; hemimethylated hairpin 3016, 0.80; lower band: Lipofectin alone 0.46, control hairpin
3060, 0.76; hemimethylated hairpin 3016, 0.56. C, bisulfite
analysis of the first exon of the MyoD gene. DNA prepared
from either A459 cells treated with the hairpin inhibitor (3016) or a
control inhibitor were subjected to bisulfite treatment and the genomic
region bearing 41 CpG sites in the first exon of the MyoD
gene was amplified by polymerase chain reaction using the primers
indicated under "Experimental Procedures" and sequenced. Eight
clones were sequenced per DNA sample. The first line
indicates the different CpG sites in the MyoD fragment. The percentage
of methylated cytosines per site in the 8 clones analyzed per treatment
were determined and are presented as different shadings of the
circles representing each of the sites as indicated.
DISCUSSION
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FOOTNOTES |
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* This work was supported in part by a grant from the Medical Research Council (Canada) (to M. S.), a research contract with Hybridon Inc., and MethylGene Inc.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.
Supported by a postdoctoral fellowship from the Medical Research
Council (Canada).
§ To whom correspondence should be addressed. Tel.: 514-398-7107; Fax: 514-398-6690; E-mail: mszyf{at}pharma.mcgill.ca.
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ABBREVIATIONS |
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The abbreviations used are: DNA MeTase, DNA methyltransferase; PAGE, polyacrylamide gel electrophoresis; AdoMet, S- adenosylmethionine.
2 A. D. Slack, N. Cervoni, M. Pinard, and M. Szyf, unpublished observations.
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REFERENCES |
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