From the Department of Chemistry and Biochemistry and
Program in Biochemistry and Molecular Biology, University of
California, Santa Barbara, California 93106, the § Shanghai
Institute of Digestive Diseases, Shanghai, China, and
¶ EpiGenX Pharmaceuticals, Pacific Technology Center, Santa
Barbara California, 93111
Received for publication, September 25, 2002, and in revised form, November 25, 2002
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ABSTRACT |
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The major DNA cytosine methyltransferase isoform
in mouse erythroleukemia cells, Dnmt1, exhibits potent dead-end
inhibition with a single-stranded nucleic acid by binding to an
allosteric site on the enzyme. The previously reported substrate
inhibition with double-stranded substrates also involves binding to an
allosteric site. Thus, both forms of inhibition
involve ternary enzyme-DNA-DNA complexes. The inhibition potency
of the single-stranded nucleic acid is determined by the sequence,
length, and most appreciably the presence of a single 5-methylcytosine
residue. A single-stranded phosphorothioate derivative inhibits
DNA methylation activity in nuclear extracts. Mouse erythroleukemia
cells treated with the phosphorothioate inhibitor show a significant
decrease in global genomic methylation levels. Inhibitor treatment of
human colon cancer cells causes demethylation of the
p16 tumor suppressor gene and subsequent p16
re-expression. Allosteric inhibitors of mammalian DNA cytosine
methyltransferases, representing a new class of molecules with
potential therapeutic applications, may be used to elucidate novel
epigenetic mechanisms that control development.
The patterns of DNA cytosine methylation in mammals evolve
throughout development (1). This essential process regulates imprinted
genes, X chromosome inactivation, the inactivation of repetitive
elements, and the expression of tissue-specific genes (2, 3).
Methylated DNA is recognized by several proteins and assembled into
transcriptionally silent chromatin structures. Gene regulatory regions,
including normally undermethylated CpG islands, become hypermethylated
with age and in tumors, thereby repressing the expression of essential
genes (2, 4, 5). Changes in DNA methylation and
AdoMet-dependent DNA cytosine methyltransferase (DCMTase)1 activity appear
early in tumorigenesis. These and other observations have motivated and
continue to motivate the development of DCMTase inhibitors as potential
"epigenetically based" drugs (2, 6, 7). Thus, antisense
oligonucleotides that interfere with DCMTase expression inhibit tumor
formation (8), and the cancer drug 5-aza-deoxycytidine (5AC;
decitabine) functions by inhibiting DCMTase (9, 10). 5AC virtually
abolished adenoma formation in mice genetically prone to colon tumors
(11). 5AC is a clinically administered mechanism-based inhibitor of
DCMTase. Unfortunately, it may be too carcinogenic and mutagenic for
most applications (10). Targeting DCMTase function with novel
inhibitors has great therapeutic potential, and novel regulators could
help elucidate the aspects of epigenetic control that occur throughout
development (2, 6, 12).
The major eukaryotic DCMTase, Dnmt1, has been cloned and sequenced from
at least five animal sources: mouse, human, chicken, frog, and sea
urchin (13-17). Several variants have been identified that arise from
separate gene loci and alternatively spliced mRNAs (1, 3). Dnmt1 is
the largest DCMTase, with a molecular mass of 184 kDa. The smaller
C-terminal domain shares sequence homology with all DCMTases and
contains all or the majority of the residues required for catalysis.
Mammalian methylation occurs predominately within the context of the
CpG dinucleotide. The N-terminal domain contains a phosphorylation site
(18) as well as zinc (19), nucleic acid (20-23), and
protein-binding elements (24). The large N-terminal domain is likely to
play an active role in the complex interplay with several proteins
including histone deacetylases and other Dnmts, leading to the active
restructuring of chromatin (1). A previous report postulating such
protein-protein interactions proposed that the substrate inhibition of
Dnmt1 at high DNA concentrations results from the loss of activating
Dnmt1-Dnmt1 interactions with adjacent enzyme molecules on the same DNA
scaffold (25).
Early evidence indicated that Dnmt1 binds a second DNA molecule at an
unidentified allosteric site (20, 21) and that N-terminal domain-derived peptides bind nucleic acids (26). These interactions recently have been characterized in detail (22, 27-29). The N-terminal domain comprising the first 501 amino acids represses the methylation reaction of the human Dnmt1; this repression is relieved upon binding
to methylated DNA (23). A similar stimulation was reported for murine
Dnmt1 upon the addition of fully methylated double-stranded DNA, again
leading to the proposal that the enzyme forms a ternary enzyme-DNA-DNA
complex (30). Another form of allosteric Dnmt1 activation, in this
circumstance involving single-stranded substrates with the potential of
forming transient duplexes, has been described for oligonucleotides
containing 5-methylcytosine adjacent to the CpG site that undergoes
methylation (31, 32). Many of these nucleic acid-mediated effects
appear to depend on the nature of the DNA substrate upon which the
enzyme is initially positioned. For example, although
poly(dI·dC) and hemimethylated
(CGG/CCG)12, a 36-bp fragment containing the triplet repeat
sequence in which expansion in the FMR1 gene causes
fragile-X syndrome, show complex kinetics consistent with DNA binding
to an allosteric site, no such behavior is observed with unmethylated
(CGG/CCG)12 (22, 23). The biological role most
frequently invoked for allosteric regulation of Dnmt1 via DNA binding
is in the context of "methylation spreading," a process leading to
de novo DNA methylation of previously ummethylated regions
(30, 34-36). This bidirectional spreading, which occurs in newly
integrated viral DNA (36), in gene regulatory sequences during X
chromosome inactivation (37), and during cellular immortalization (38),
in transposed DNA sequences (39), may be a threat to the success of
gene therapy (40).
Our interests were to address the mechanism of the well known substrate
inhibition that underlies much of the complex kinetics reported for
Dnmt1 and to further characterize the interactions of the enzyme with
several single-stranded DNA molecules that were previously shown to be
poor substrates for the enzyme (41). The enzyme binds these poor
substrates with affinities comparable with substrates that are readily
methylated (21), and we hypothesized that they might act as dead-end
inhibitors. A previous report of nucleic acids that bind Dnmt1 but are
poor substrates showed that various single- and double-stranded nucleic
acids can act as inhibitors (42). Here we report the mechanism of
action of one such single-stranded inhibitor and demonstrate its
ability to inhibit methylation in vitro.
Materials--
S-Adenosyl-L-[methyl-3H]methionine
(75 Ci/mmol, 1 mCi/ ml = 37 GBq) was purchased from
Amersham Biosciences. Unlabeled AdoMet (Sigma) was further
purified as described (43). Routinely, 125 µM AdoMet
stocks were prepared at a specific activity of 5.8 × 103 cpm/pmol. Two lots of poly(dI·dC-dI·dC) were
purchased from Amersham Biosciences, with an average length of 6250 and
5000 base pairs. DE81 filters were purchased from Whatman.
LipofectAMINE was purchased from Invitrogen. Other standard chemicals
and reagents were purchased from Sigma or Fisher Scientific.
Oligonucleotides were synthesized by Research Genetics (Huntsville, AL)
and HPLC-purified on a Dynamax PureDNA column (Rainin Instrument
Co.) according to the manufacturer's specifications. Oligonucleotides
were stored in 10 mM Tris, pH 8.0, 1 mM EDTA.
The following sequences were prepared (GC-box pMET, LC2,
and the antisense sequence (8) have a phosphorothioate backbone; all
others contain a deoxyribose backbone; M superscript denotes
5-methylcytosine; the numbers refer to nucleotide length; the
recognition CpG dinucleotides are underlined and bold): 30, GC-boxb,
d(CTGGATCCTTGCCCCGCCCCTTGAATTCCC); 30, GC-boxa,
d(GGGAATTCAAGGGGCGGGGCAAGGATCCAG); 30, LC2,
d(CTGGATCCTTGCCAAAACCCTTGAATTCCC); 30, GC-boxbMET,
d(CTGGATCCTTGCCCMCGCCCCTTGAATTCCC);
30, GC-boxpMET, d(CTGGATCCTTGCCC
MCGCCCCTTGAATTCCC); 50, GC-boxcMET, d(CCTACCCACC-(GC-box
bMET)-AACCCTCCAC); 22, GC-boxdMET,
d(ATCCTTGCCCMCGCCCCTTGAAT);
14, GC-boxeMET, d(TTGCCC
MCGCCCCTT); 30, CRE,
d(GGGAATTCAAATGAMCGTCAAAAGGATCCAG);
20, antisense, d(TCTATTTGAGTCTGCCATTT).
Concentrations were determined using calculated coefficients, and the
annealing of GC-boxa/b was as described previously (41).
Dnmt1 was isolated from mouse erythroleukemia cells (MEL cells) as
described (41).
Methyltransferase Assays--
Filter binding assays monitored
the incorporation of tritium-labeled methyl groups into DNA. Reaction
and dilution buffers and filter processing were described previously
(20). Incubations were at 37 °C for 60 min. Each point represents
the average of at least two reactions.
Substrate Inhibition--
Initial velocity data were collected
with a 6250-base pair poly(dI·dC) and a 30-base pair
GC-boxa/b substrate. The poly(dI·dC) concentrations were
~2.0, 4.0, 8.0, 16, 35, 80, 160, 250, 400, 700, and 1000 picomolar in
duplex DNA; Dnmt1 was 3.0 nM. The GC-boxa/b
substrate concentrations were (0.20, 0.40, 1.0, 2.0, 4.0, 8.0, 15, 23, and 35 µM); Dnmt1 was 100 nM.
Inhibition with Single-stranded Oligonucleotides--
Initial
velocity data were collected with poly(dI·dC), AdoMet,
GC-boxb, and GC-boxbMET at the concentrations
indicated in Fig. 2 legend. Dnmt1 was either 3 or 4 nM (see
Fig. 2 legend), AdoMet was 10 µM when added as the
nonchanging substrate, and poly(dI·dC) was 50 pM when
added as the nonchanging substrate. IC50 determinations
with GC-boxbMET, GC-boxpMET,
GC-boxcMET, GC-boxdMET, GC-boxeMET,
and CRE used 4 nM Dnmt1 and 50 pM
poly(dI·dC). Oligonucleotide concentrations ranged between 5-fold
lower and 5-fold higher than the IC50.
Nuclear Extract Assays--
MEL nuclear extracts were prepared
as described previously (41). Freshly made nuclear extract (2 µl) was
combined with 12.5 µM tritiated AdoMet, 1 nM
poly(dI·dC), and varying concentrations of oligonucleotides in a
volume of 20 µl at 37 °C. The reaction was stopped after 60 min,
and label incorporation into DNA was determined as described (41).
Mouse Erythroleukemia Cell Studies--
MEL cells, prepared as
described (41) to a density of 106/ml, were treated with
inhibitors (7.7 µM GC-boxpMET, 1.5 µM 5AC, or 18 µM antisense oligonucleotide)
along with LipofectAMINE as described by the manufacturer. The mock
treatment used TE (Tris, pH 8.0 (10 mM), and EDTA, 1 mM) in place of any inhibitor. The inhibitors or the mock
were added only at the initiation of the experiment, and genomic DNA
was isolated after 72 and 110 h of cell culture. 5-Methylcytosine
content was determined as described (44). Briefly, MspI
endonuclease was used to digest the genomic DNA, and the samples were
then treated sequentially with calf intestinal alkaline phosphatase, T4
polynucleotide kinase (and [ Human Colon Cancer Cell Studies--
HT29 cells were plated at
50% confluence in six-well plates and treated with 5AC at 1 µM or LipofectAMINE-transfected with either 10 µM GC-boxbMET or LC2 control oligonucleotides
for 48-72 h. The LC2 control is identical to GC-boxbMET
with the four central bases converted to adenines. DNA was isolated using the Qiagen blood and cell culture DNA kit (Qiagen) according to
the manufacturer's instructions. Methyl-specific PCR (MSP) was
performed as described previously (45) using primers described therein.
Peripheral blood lymphocytes were used as a positive control for
unmethylated p16, and peripheral blood lymphocyte DNA,
methylated in vitro using SssI methylase (New
England Biolabs) according to the manufacturer's instructions, was
used as a positive control for methylated p16. RNA was
isolated using Trizol (Invitrogen). cDNA was prepared from 1 µg
of RNA using a Superscript II reverse transcription system with random
hexamers as primers (Invitrogen). PCR was performed using primers for
p16 designed to cross a splice junction in the gene
(GenBankTM accession number L27211), 5'-ATC ATC AGT
CAC CGA AGG TC-3' (sense) and 5'-CCA CAT GAA TGT GCG CTT AG-3'
(antisense), on 1 µl of cDNA product. The reaction was initiated
with a 3-min incubation at 94 °C followed by 35 amplification cycles
(94 °C for 30 s, 58 °C for1 min, 72 °C for 1 min) and a
final 10 min extension step of 10 min yielding a 355-bp product
following electrophoresis on a 1.5% agarose gel. The gel was stained
with ethidium bromide and photographed. Expression of The original observation by Linn and co-workers (25) that
hemimethylated duplex plasmid DNA and single-stranded DNA are inhibitory at high concentrations was explained with an interesting model in which Dnmt1 associates with itself upon the DNA scaffold at
low ratios of DNA to enzyme, thereby activating the enzyme. Monomeric
enzyme-DNA complexes are stabilized with excess substrate, thus leading
to a loss of the activated form. An alternative explanation for this
behavior at high DNA concentrations is the formation of an inhibitory
ternary complex involving the enzyme and two or more DNA molecules.
Although recent reports provide data consistent with this alternative
explanation (23, 30), we sought to provide a simple probe of the
underlying mechanism. Fig. 1 shows our
results with a large multi-site 6250-bp poly(dI·dC) substrate and a
short 30-bp GC-boxa/b duplex containing a single CpG site.
The GC-boxa/b duplex is an excellent substrate for Dnmt1
and contains an Sp1 transcription factor recognition element (41). We
and others have demonstrated that poly(dI·dC) shows inhibition at
high DNA concentrations (20, 23, 46), which is revealed by the data in
Fig. 1. The relative velocities versus
S/Km plots conveniently normalize the data between
the two diverse DNA substrates. The short 30-bp duplex nearly matches
the footprint of the Dnmt1-DNA complex (20), and we reasoned that this
substrate would be precluded from stabilizing the types of complexes
proposed by Linn and coworkers (25). As shown in Fig. 1, the
GC-boxa/b duplex shows the same form of substrate
inhibition as the much larger poly(dI·dC). The poorer affinity
observed with the 30-bp duplex limited our ability to take the
S/Km ratio as high as with the poly(dI·dC);
nevertheless, both sets of data are nicely fit to a standard equation
for substrate inhibition, invoking formation of an inhibitory ternary
enzyme-DNA-DNA complex (47).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32P]ATP), and P1
nuclease. The 32P end-labeled cytosines, either
5-mCMP or CMP within the CpG of the MspI ends,
were separated on cellulose thin layer chromatography plates. A
PhophorImager (Amersham Biosciences) was used to visualize and quantify
the percent 5-methylcytosine. Each reported result is the average of at
least two independent determinations.
-actin was
used as a standard for RNA integrity and equal gel loading. Primers for
-actin were 5'-GGA GTC CTG TGG CAT CCA CG-3' (sense) and 5'-CTA GAA
GCA TTT GCG GTG GA-3' (antisense). Amplification was the same as p16
with the exception of using an annealing temperature of 60 °C and 27 amplification cycles.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
DNA substrate inhibition of Dnmt1.
A, initial velocity plots of DNA methyltransferase activity
with poly(dI·dC:dI·dC) (6250 bp) and GC-boxa/b (30 bp).
For a direct comparison, the data are expressed in an
S/Km ratio (for DNA). The
poly(dI·dC:dI·dC) concentrations were 2.0, 4.0, 8.0, 16, 35, 80, 160, 250, 400, 700, and 1000 pM. Dnmt1 concentration was
3.0 nM. B shows data in which
GC-boxa/b (0.20, 0.40, 1.0, 2.0, 4.0, 8.0, 15, 23, and 35 µM) was used with 100 nM Dnmt1. The lines
represent the best fit to the data using a model of simple substrate
inhibition (formation of an inactive, ternary
enzyme-substrate-substrate complex).
Our results provide compelling support for a mechanism involving the binding of a second double-stranded DNA molecule to an allosteric site on Dnmt1. The observed inhibition with the short duplex DNA (Fig. 1) is unlikely to come from the preferential stabilization of monomeric enzyme forms, because these short substrates were designed to be incapable of stabilizing such complexes under any enzyme concentrations. In this circumstance limited to unmethylated duplexes (poly(dI·dC) or GC-boxa/b), the binding of a second DNA molecule is inhibitory. We recently confirmed this result with unmethylated DNA and another assay, showing that the ternary enzyme-DNA-DNA complexes dramatically decrease the processive action of the enzyme on multi-site substrates by increasing the off-rate from the substrate DNA.2 Such an increased off-rate could have the same effect with the single-site substrate (GC-boxa/b) if the off-rate and methylation rates are comparable. In contrast to these inhibitory allosteric effects, we and others (Ref. 30 and footnote 2) have shown that methylated DNA activates Dnmt1. This occurs in trans, with 5-methylcytosine attached to DNA other than that being methylated, and in cis, when introduced on the same molecule at positions adjacent to the target methylation sequence (CpG) (31, 48)).3 It remains unclear whether the activation and inhibition effects are mediated by nucleic acid binding to the same allosteric site.
Dnmt1 shows good activity with single-stranded plasmid-derived
substrates (25) and single-stranded oligonucleotides (Refs. 31 and 34,
and footnote 3); we previously determined this with single-stranded
versions of CRE, a cis-regulatory transcriptional element
(41). Some single-stranded oligonucleotides are proposed to form
transient duplexes that are then acted upon by the enzyme (31); the CRE
and GC-box-derived single strands used in our study show no such
behavior (41). In contrast to the CRE-derived strands, single-stranded
substrates mimicking the GC-box element, showed no detectable activity
(41) despite the ability of the enzyme to bind such sequences with good
affinity (21). We therefore speculated that the single-stranded GC-box
oligonucleotides could act as dead-end inhibitors of the enzyme. We
submitted GC-boxb single-stranded DNA to a detailed
inhibition analysis; the double reciprocal plots are shown in Fig.
2A. The pattern of lines
intersects far to the left of the y axis and is best fit by
a standard equation for noncompetitive inhibition. The inhibition
constants were determined to be Kis = 3.6 ± 1.5 µM and Kii = 6.8 ± 1.2 µM (49). Kii describes the
inhibition constant for the enzyme-substrate complex, and Kis the inhibition constant for the free enzyme.
The Kii/Kis ratio is a
measure of which binding event is preferred (49), and in this case, its
value of 1.9 suggests that this inhibitor slightly favors addition to
the free enzyme over the enzyme-DNA complex. Thus, the pattern shows
that the free enzyme can bind to single-stranded GC-boxb,
as can the binary enzyme-poly(dI·dC) complex. In both cases the
enzyme is inhibited. The latter ternary complex requires that GC-boxb binds to an allosteric site.
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Dead-end inhibition with a CpG methylated homolog of GC-boxb, GC-boxbMET, also exhibited noncompetitive inhibition (Fig. 2B). Remarkably, a single 5-mC substitution reduces the inhibition constant associated with the allosteric site 340-fold, as Kii = 20 ± 3 nM. The pattern of lines is visibly less convergent than in Fig. 2A, and Kii/Kis = 0.57. The unmethylated and methylated inhibitors have distinct partitioning preferences as suggested by the changes in binding affinity and Kii/Kis. These results strongly suggest that GC-boxbMET and poly(dI·dC) can and do bind preferentially to distinct sites on Dnmt1, with the single methyl moiety driving that preference in the favor of the allosteric site.
The initial velocity patterns obtained by varying AdoMet and GC-boxbMET concentrations were best fit to a standard equation for competitive inhibition (Fig. 2C). The intersection of the fit lines on the 1/velocity axis, Kis = 25 ± 10 nM, suggests that GC-boxbMET and AdoMet bind competitively to the same poly(dI·dC)-bound form of the enzyme but not necessarily to the same site on the enzyme. The inhibition constants determined for GC-boxbMET at the proposed allosteric site, Kis in Fig. 2C and Kii in Fig. 2B, are in good agreement at about 30 nM. In the absence of poly(dI·dC), GC-boxbMET binds free enzyme with a dissociation constant of 880 nM (21). Therefore, poly(dI·dC) stabilizes GC-boxbMET binding 29-fold.
The binding of these duplex and single-stranded nucleic acids to a site on Dnmt1 distinct from the substrate-binding site is intriguing. The evidence for the existence of such an allosteric site is compelling: (i) the substrate inhibition described in Fig. 1 for both long and short substrates is best explained by the binding of a second DNA molecule; (ii) noncompetitive, dead-end inhibition by single-stranded oligonucleotides (Fig. 2, A and B) was dramatically enhanced when the inhibitor contained 5-methylcytosine; (iii) no inhibition with either GC-boxb or GC-boxbMET was observed with two bacterial DNA cytosine methyltransferases, M.HhaI and M.SssI (data not shown); (iv) previous product inhibition studies support a Dnmt1-DNA-DNA ternary complex (20, 22, 23, 25); (v) DNA concentrations 10 times higher than Km produce a second, less mobile band by gel mobility shift assays (20); (vi) short peptides from the N terminus of Dnmt1 have primary sequence similarity to zinc fingers and bind double-stranded DNA, although single-stranded DNA was not tested (24).
Allosteric enzyme regulators provide a novel approach to alter enzyme function and thus to introduce new strategies for drug design (50). Because GC-boxbMET is a potent allosteric inhibitor, we sought to define those features that are essential for its activity. Derivatives of GC-boxbMET were tested to determine how length, base, and backbone composition affect Dnmt1 inhibition (Table I). The IC50, defined as that concentration of inhibitor that causes 50% inhibition of the methylation reaction with poly(dI·dC), increased 2-fold in going from 30 to 50 nucleotides, being 15 and 30 nM, respectively. Significantly weaker binding was observed with 22 and 14 nucleotide lengths. Thus the enzyme appears to interact with at least 30 nucleotides through this allosteric site. Changing the backbone to a phosphorothioate, GC-boxpMET, decreased the IC50 from 15 nM for the comparable 30-nucleotide GC-boxbMET to 5 nM. This 3-fold better binding could derive from various effects, including altered electrostatics to subtle changes in the allowed conformations of the oligonucleotides. Sequence specificity was demonstrated with CREaMET, a relatively adenine/thymine-rich element compared with the GC-box inhibitors. CREaMET was at least 60-fold less inhibitory than our most potent inhibitor, GC-box pMET (Table I), and showed competitive inhibition against poly(dI·dC) (data not shown). We suggest that CREaMET and the substrate DNA (poly(dI·dC)) bind the same form of the enzyme, a binding that most likely occurs at the active site. This is supported by our observations that the nonmethylated version of CREaMET (CRE a) is an excellent substrate for Dnmt1 (41) and that CREaMET shows competitive inhibition.
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Our data provide the first evidence that an allosteric site on Dnmt1 shows DNA sequence preferences, which almost certainly opens up novel regulatory pathways. The data provided here and by others show that one or more allosteric site(s), most likely residing within the N-terminal domain, can bind double- or single-stranded nucleic acids. This binding is highly dependent on methylation status and DNA sequence, as well as on length and backbone composition. Although much needs to be learned about the site within the protein, its interactions with nucleic acids, and its biological role, there can be no question that the function of Dnmt1 is regulated by an allosteric mechanism.
Novel in vitro modulators of DCMTase activity could possibly
alter genomic methylation patterns, alter gene expression and control
the unrestricted proliferation of cancerous cells (3, 6, 51). The
epigenetic inactivation of genes is as important a driving force in
tumorigenesis as the inactivation of genes by mutation (52). Epigenetic
transcriptional repression has been demonstrated in diverse tumor types
including tumor suppressor genes, DNA repair genes, cell-cycle genes,
and genes involved in invasion and metastasis (5, 12, 52). The
re-expression of such genes in tumor cells leads to cell growth
suppression and altered sensitivity to existing cancer therapies.
However, despite encouraging results with available approaches to alter cellular DNA methylation, there is clearly a need for potent Dnmt1 inhibitors with improved drug characteristics (3, 6, 51). Based on the
nanomolar potency of GC-boxpMET, its novel mechanism of
inhibition, and its phosphorothioate backbone, we sought to determine
whether this compound could alter methylation in nuclear extracts as
well as in cells. We tested the effectiveness of GC-boxpMET
in nuclear extracts from MEL cells (41). Potent inhibition of DNA
methylation was observed with an IC50 of 45 nM
(Fig. 3A). A direct comparison
with our results obtained with purified Dnmt1 (Table I) is complicated
by several factors, such as Dnmt1 concentration and allosteric site
accessibility. If significant levels of other DCMTase family members
exist in MEL nuclear extracts (1, 3), then the inhibitor appears to
regulate them also, because methylation activity was potently decreased
to background levels.
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To test the efficacy of the inhibitor in intact cells, we treated MEL
cells with a mock inhibitor solution, 1.5 µM 5AC, 7.7 µM GC-boxpMET, or 18 µM of an
antisense to Dnmt1 mRNA. The antisense oligonucleotide was
previously shown to alter cellular DNA methylation levels and is in
Phase II clinical trials for the treatment of cancer (8). Genomic DNA
was isolated after 72 and 110 h of cell culture, and the
percentage of 5-mC within CpG sites was used to assess
genomic methylation levels (44). The 5-mC content decreased
in a time-dependent manner in all treatment cases (Fig.
3B). A decrease in 5-mC was evident after
110 h using 5AC (Fig. 3B). Likewise,
GC-boxpMET was effective in reducing the 5-mC
content, whereas the Dnmt1 antisense oligonucleotide was the least
effective. Although it was not as effective as 5AC under these
conditions, GC-boxpMET did not appear to damage the cells;
5AC did damage the cells, resulting in significant degradation
of cellular DNA. These results on genomic methylation levels were
complemented by our characterization of the impact of the inhibitor on
the methylation of a specific locus, in which inappropriate methylation
was previously shown to cause gene expression changes leading to
tumorigenesis. The HT29 human colon cell line was used to examine the
ability of LipofectAMINE-transfected GC-boxbMET to cause
gene-specific demethylation of the tumor suppressor gene,
p16 (12, 53). The aberrant methylation of the CpG island associated with the p16 gene occurs in diverse human cancers
with frequencies up to 48% (lymphoma), 39% (pancreatic), and 37%
(colon) (53). Reversal of this methylation with 5AC treatment results in re-expression of p16. MSP was used to determine the methylation status of individual CpG sites (54). Treatment of the HT29 cells with
5AC results in demethylation (Fig.
4A, lane 4) and
expression of p16 (Fig. 4B, lane 3) and was used
as a positive control. A similar experiment performed with
LipofectAMINE-transfected GC-boxbMET showed specific
demethylation of p16 (Fig. 4A, lane
3) and re-expression of the gene (Fig. 4B, lane
4), whereas a control oligonucleotide (LC2, see "Experimental
Procedures") had no effect on p16-specific demethylation in HT29 (Fig
4, A, lane 2 and B, lane
5). The LC2 control results argue persuasively for a specific
effect deriving from the inhibitor on the cellular processes leading to
the p16 CpG island methylation.
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In conclusion, we and others have previously identified low affinity
interactions between Dnmt1 and various nucleic acids (22, 23, 25, 30,
41, 48, 55), effects that predominately involve the active site of the
enzyme. Although the underlying mechanisms remain obscure in most of
these cases, it is clear that they are distinct from the inhibition
described in this report. Cell-active, tight binding, allosteric Dnmt1
inhibitors may form the basis of a new class of cancer drugs. Such
therapies are likely to involve treatment with other epigenetically
based drugs, such as histone deacetylase inhibitors, and to complement
chemotherapies that are vulnerable to methylation-dependent
resistance mechanisms (33). Although oncogenic transformation
has been reversed with 5AC, allosteric inhibitors can perhaps separate
the beneficial effects of gene reactivation from the known cytotoxic
effects of 5AC.
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed: Dept. of Chemistry
and Biochemistry, University of California, Santa Barbara, CA 93106. E-mail: reich@mail.chem.ucsb.edu.
Published, JBC Papers in Press, December 10, 2002, DOI 10.1074/jbc.M209839200
2 Z. Svedruzic and N. O. Reich, submitted for publication.
3 B. Aubol and N. O. Reich, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: DCMTase, DNA cytosine methyltransferase; 5AC, 5-aza-deoxycytidine (decitabine); MEL, mouse erythroleukemia cells; AdoMet, S-adenosylmethionine; CRE, cAMP-response element; MSP, methyl-specific PCR; 5-mC, 5-methylcytosine residue.
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