U.S. patent application number 09/824308 was filed with the patent office on 2001-11-15 for specific inhibitors of dna methyltransferase.
Invention is credited to Bigey, Pascal, Szyf, Moshe.
Application Number | 20010041337 09/824308 |
Document ID | / |
Family ID | 26750453 |
Filed Date | 2001-11-15 |
United States Patent
Application |
20010041337 |
Kind Code |
A1 |
Szyf, Moshe ; et
al. |
November 15, 2001 |
Specific inhibitors of DNA methyltransferase
Abstract
The invention provides novel mechanism-based inhibitors of DNA
methyltransferase enzyme, and diagnostic and therapeutic uses for
the same. The novel inhibitors according to the invention form
stable, noncovalent complexes with DNA methyltransferase enzyme in
a manner which is independent of S-adenosylmethionine.
Inventors: |
Szyf, Moshe; (Cote St. Luc,
CA) ; Bigey, Pascal; (Clermont-Ferrand, FR) |
Correspondence
Address: |
HALE AND DORR, LLP
60 STATE STREET
BOSTON
MA
02109
|
Family ID: |
26750453 |
Appl. No.: |
09/824308 |
Filed: |
April 2, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09824308 |
Apr 2, 2001 |
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09206866 |
Dec 8, 1998 |
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6268137 |
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09824308 |
Apr 2, 2001 |
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09194284 |
Jun 10, 1999 |
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09194284 |
Jun 10, 1999 |
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PCT/IB97/00879 |
May 22, 1997 |
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PCT/IB97/00879 |
May 22, 1997 |
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08653954 |
May 22, 1996 |
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60069812 |
Dec 17, 1997 |
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Current U.S.
Class: |
435/6.13 ;
435/6.15; 514/44A; 536/23.2 |
Current CPC
Class: |
C12N 2310/334 20130101;
C12N 2310/53 20130101; C12N 2310/315 20130101; C12N 2310/13
20130101; C07H 21/00 20130101; C12N 15/1137 20130101; A61K 38/00
20130101 |
Class at
Publication: |
435/6 ; 514/44;
536/23.2 |
International
Class: |
C12Q 001/68; C07H
021/04; A61K 048/00 |
Claims
What is claimed is:
1. An inhibitor of DNA methytransferase enzyme having the general
structure: 6wherein each N is independently any nucleotide, n is a
number from 0-20, C is 5-methylcytidine, G is guanidine, y is a
number from 0-20, L is a linker, each D is a nucleotide that is
complementary to an N such that Watson-Crick base pairing takes
place between that D and the N such that the N.sub.n--C--G--N.sub.y
and the D.sub.n--G--B--D.sub.y form a double helix, B is cytosine,
inosine, uridine, 5-bromocytosine or 5-fluorocytosine, orabasic
deoxyribose, the linkage between B and G is a phosphorothioate or
phosphorodithioate linkage, dotted lines between nucleotides
represent hydrogen bonding between the nucleotides, and the total
number of nucleotides ranges from about 10 to about 50.
2. The inhibitor of DNA methytransferase enzyme according to claim
1, wherein the inhibitor is labeled.
3. The inhibitor of DNA methytransferase enzyme according to claim
1, wherein L is a nucleoside or an oligonucleotide region having
from 2 to about 10 nucleotides.
4. The inhibitor of DNA methytransferase enzyme according to claim
3 comprising at least one internucleoside linkage selected from the
group consisting of phosphodiester, phosphotriester,
phosphorothioate, or phosphoramidate linkages, or combinations
thereof.
5. The inhibitor of DNA methytransferase enzyme according to claim
3 comprising at least one phosphorothioate internucleoside
linkage.
6. The inhibitor of DNA methyltransferase enzyme according to claim
1, having the sequence selected from the group consisting of SEQ.
ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID.
NO. 5, SEQ. ID. NO. 6, SEQ. ID. NO. 7, SEQ. ID. NO. 8, SEQ. ID. NO.
9, SEQ. ID. NO. 10, SEQ. ID. NO. 11, SEQ. ID. NO. 12, SEQ. ID. NO.
13, SEQ. ID. NO. 14, SEQ. ID. NO. 15, SEQ. ID. NO. 16, SEQ. ID. NO.
17, SEQ. ID. NO. 18, SEQ. ID. NO. 19, SEQ. ID. NO. 20, SEQ. ID. NO.
21, SEQ. ID. NO. 22, SEQ. ID. NO. 23, SEQ. ID. NO. 24, SEQ. ID. NO.
25, SEQ. ID. NO. 26, SEQ. ID. NO. 27, SEQ. ID. NO. 28, SEQ. ID. NO.
29, SEQ. ID. NO. 30, SEQ. ID. NO. 31, SEQ. ID. NO. 32, SEQ. ID. NO.
33, SEQ. ID. NO. 34, SEQ. ID. NO. 35, SEQ. ID. NO. 36, SEQ. ID. NO.
37, SEQ. ID. NO. 38, SEQ. ID. NO. 39, SEQ. ID. NO. 40 and SEQ. ID.
NO. 41, wherein C is cytidine, T is thymidine, A is adenosine, G is
guanine, m is a methyl group at the 5-position of cytosine, B is
cytosine, inosine, uridine, 5-bromocytidine, or 5-fluorouridine, X
is any base and F is 5-fluorocytosine.
7. An inhibitor of DNA methytransferase enzyme having the general
structure 7wherein each N is independently any nucleotide, n is a
number from 0-20, C is 5-methylcytidine, G is guanidine, y is a
number from 0-20, L is a linker, each D is a nucleotide that is
complementary to an N such that Watson-Crick base pairing takes
place between that D and the N such that the N.sub.n--C--G--N.sub.y
and the D.sub.n--G--B--D.sub.y form a double helix, B is cytosine,
inosine, uridine, 5-bromocytosine, abasic deoxyribose, or
5-fluorocytosine, dotted lines between nucleotides represent
hydrogen bonding between the nucleotides, B and G are linked by a
phosphorothioate or phosphorodithioate linkage and the total number
of nucleotides ranges from about 10 to about 50, X is an antisense
oligonucleotide of from about 10 to about 50 nucleotides in length,
which is complementary to a portion of an RNA encoding DNA MTase
enzyme, and L can optionally be X.
8. A diagnostic method for determining whether a particular sample
of cells is cancerous, the method comprising preparing an extract
from the cells in the cell sample, adding a labeled inhibitor
according to claim 2, measuring the extent of formation of a
complex between the labeled inhibitor and DNA MTase enzyme,
normalizing the level of such complex formation to the number of
cells represented in the sample to obtain a normalized complex
formation value, and comparing the normalized complex formation
value to a normalized complex formation value for non-cancerous
and/ or cancerous cell samples.
9. The method according to claim 8, wherein the extract is a
nuclear extract.
10. A method for inhibiting tumorigenesis in an animal, including a
human, comprising administering to the animal, which has cancer
cells present in its body, a therapeutically effective amount of an
inhibitor according to claim 1 for a therapeutically effective
period of time.
Description
[0001] This is a continuation-in-part of U.S. provisional
application No. 60/069,812, filed Dec. 17, 1997, a
continuation-in-part of U.S. Ser. No. 09/194,284 (U.S. National
Phase application of PCT/IB97/00879, filed November 23, 1998), and
of PCT/IB97/00879, filed May 22, 1997, which is a
continuation-in-part of U.S. Ser. No. 08/653,954, filed May 22,
1996.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to modulation of gene expression. In
particular, the invention relates to modulation of gene expression
of the gene encoding DNA methyltransferase, and to modulation of
gene expression that is regulated by the enzyme DNA
methyltransferase.
[0004] 2. Summary of the Related Art
[0005] Modulation of gene expression has become an increasingly
important approach to understanding various cellular processes and
their underlying biochemical pathways. Such understanding enriches
scientific knowledge and helps lead to new discoveries of how
aberrancies in such pathways can lead to serious disease states.
Ultimately, such discoveries can lead to the development of
effective therapeutic treatments for these diseases.
[0006] One type of cellular process that is of particular interest
is how the cell regulates the expression of its genes. Aberrant
gene expression appears to be responsible for a wide variety of
inherited genetic disorders, and has also been implicated in
numerous cancers and other diseases. Regulation of gene expression
is a complex process, and many aspects of this process remain to be
understood. One of the mysteries of this process resides in the
fact that while the genetic information is the same in all tissues
that constitute a multicellular organism, the expression of
functions encoded by the genome varies significantly in different
tissues.
[0007] In some cases, tissue-specific transcription factors are
known to play a role in this phenomenon. (See Maniatis et al.,
Science 236:1237-1245 (1987); Ingarham et al., Annual Review of
Physiology 52:773-791 (1990). However, several important cases
exist that cannot be readily explained by the action of
transcription factors alone. For example, Midgeon, Trends Genet.
10:230-235 (1994), teaches that X-inactivation involves the
inactivation of an allele of a gene that resides on the inactive
X-chromosome, while the allele on the active X-chromosome continues
to be expressed. In addition, Peterson and Sapienza, Annu. Rev.
Genet. 27:7-31 (1993), describes "parental imprinting", where an
allele of a gene that is inherited from one parent is active and
the other allele inherited from the other parent is inactive. In
both of these cases, both alleles exist in an environment
containing the same transcription factors, yet one allele is
expressed and the other is silent. Thus, something other than
transcription factors must be involved in these phenomena.
[0008] Investigators have been probing what type of "epigenetic
information" may be involved in this additional control of the
expression pattern of the genome. Holliday, Philos. Trans. R. Soc.
Lond. B. Biol. Sci. 326:329-338 (1990) discusses the possible role
for DNA methylation in such epigenetic inheritance. DNA contains a
set of modifications that is not encoded in the genetic sequence,
but is added covalently to DNA using a different enzymatic
machinery. These modifications take the form of methylation at the
5 position of cytosine bases in CG dinucleotides. Numerous studies
have suggested that such methylation may well be involved in
regulating gene expression, but its precise role has remained
elusive. For example, Lock et al., Cell 48:39-46 (1987), raises
questions about whether the timing of hypermethylation and
X-inactivation is consistent with a causal role for methylation.
Similarly, Bartolomei et al., Genes Dev. 7:1663-1673 (1993) and
Brandeis et al., EMBO J. 12:3669-3677 (1993), disclose
timing/causation questions for the role of methylation in parental
imprinting.
[0009] Some of the shortcomings of existing studies of the role of
DNA methylation in gene expression reside in the tools that are
currently available for conducting the studies. Many studies have
employed 5-azaC to inhibit DNA methylation. However, 5-azaC is a
nucleoside analog that has multiple effects on cellular mechanisms
other than DNA methylation, thus making it difficult to interpret
data obtained from these studies. Similarly, 5-azadC forms a
mechanism based inhibitor upon integration into DNA, but it can
cause trapping of DNA methyltransferase (MTase) molecules on the
DNA, resulting in toxicities that may obscure data
interpretation.
[0010] More recently, Szyf et al., J. Biol. Chem. 267:12831-12836
(1995), discloses a more promising approach using expression of
antisense RNA complementary to the DNA MTase gene to study the
effect of methylation on cancer cells. Szyf and Von Hofe,
PCT/US94/13685 (1994), discloses the use of antisense
oligonucleotides complementary to the DNA MTase gene to inhibit
tumorigenicity. These developments have provided powerful new tools
for probing the role of methylation in numerous cellular processes.
In addition, they have provided promising new approaches for
developing therapeutic compounds that can modulate DNA methylation.
One limitation to these approaches is that their effect is not
immediate, due to the half life of DNA MTase enzyme. Thus, although
the expression of DNA MTase is modulated, residual DNA MTase enzyme
can continue to methylate DNA until such residual enzyme is
degraded. There is, therefore, a need for new inhibitors of DNA
MTase enzyme which are effective at inhibiting methylation, but
without the toxic side effects of the earlier mechanism-based
inhibitors.
BRIEF SUMMARY OF THE INVENTION
[0011] The invention provides novel inhibitors of DNA MTase enzyme
and methods for using such inhibitors as analytical and diagnostic
tools, as potentiators of transgenic plant and animal studies and
gene therapy approaches, and as potential therapeutic agents.
[0012] In a first aspect, the invention provides novel hairpin
oligonucleotide inhibitors of DNA methyltransferase (DNA MTase)
enzyme. The normal substrate for DNA MTase is a hemimethylated
double stranded DNA molecule having a CG dinucleotide opposite a
5-methyl CG dinucleotide, e.g., in a hairpin forming
oligonucleotide. Methylation occurs at the 5-position of the
cytosine base in the CG dinucleotide. The present inventors have
discovered that substitution of the CG dinucleotide with a
phosphorothioate IG, UG, 5-bromocytosineG, 5-fluorocytosineG,
abasicG or CG dinucleotide in a hairpin forming oligonucleotide
results in a powerful mechanism-based inhibitor of DNA MTase. Thus,
inhibitors according to this aspect of the invention have the
general structure: 1
[0013] wherein each N is independently any nucleotide, n is a
number from 0-20, C is 5-methylcytidine, G is guanidine, y is a
number from 0-20, L is a linker, each D is a nucleotide that is
complementary to an N such that Watson-Crick base pairing takes
place between that D and the N such that the N.sub.n--C--G--N.sub.y
and the D.sub.n--G--B--D.sub.y form a double helix, B is cytosine,
inosine, uridine, 5-bromocytosine, abasic deoxyribose, or
5-fluorocytosine, dotted lines between nucleotides represent
hydrogen bonding between the nucleotides, the linkage between G and
B is a phosphorothioate or phosphorodithioate linkage and the total
number of nucleotides ranges from about 10 to about 50. In one
preferred embodiment, L is an oligonucleotide region having from 1
to about 10 nucleotides. DNA MTase inhibitors according to this
aspect of the invention bind DNA MTase enzyme avidly in a
noncovalent manner and inhibit DNA MTase in an S-adenosylmethionine
(SAM)-independent manner.
[0014] In a second aspect, the invention provides inhibitors of DNA
MTase enzyme which also inhibit the expression of the DNA MTase
gene. Inhibitors according to this aspect of the invention have the
general structure: 2
[0015] wherein the substituents are the same as for inhibitors
according to the first aspect of the invention, except that X is an
antisense oligonucleotide of from about 10 to about 50 nucleotides
in length, which is complementary to a portion of an RNA encoding
DNA MTase enzyme, and L can optionally be X.
[0016] In a third aspect, the invention provides a diagnostic
method for determining whether a particular sample of cells is
cancerous. The method according to this aspect of the invention
comprises preparing an extract from the cells in the cell sample,
adding labeled inhibitor according to the invention, measuring the
extent of formation of a complex between the labeled inhibitor and
DNA MTase enzyme, normalizing the level of such complex formation
to the number of cells represented in the sample to obtain a
normalized complex formation value, and comparing the normalized
complex formation value to a normalized complex formation value for
non-cancerous and/ or cancerous cell samples. In a preferred
embodiment, the extract is a nuclear extract. Because cancer cells
express DNA MTase at much higher levels than do non-cancerous
cells, the comparison of the normalized complex formation values is
diagnostic for whether the cell sample is cancerous.
[0017] In a fourth aspect, the invention provides methods for
inhibiting tumorigenesis comprising administering to an animal,
including a human, inhibitors according to the invention. In the
method according to this aspect of the invention a therapeutically
effective amount of an inhibitor according to the invention is
administered for a therapeutically effective period of time to an
animal, including a human, which has cancer cells present in its
body.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a diagrammatic representation showing inhibition
of DNA MTase enzyme by certain preferred embodiments of DNA MTase
enzyme inhibitors according to the invention.
[0019] FIG. 2 are representations of autoradiographs (panels A, B
and D) and Western blots (panel C) in an experiment to identify
complex formation between DNA MTase inhibitors and DNA MTase
enzyme. Complex formation was reversed by boiling, and was
independent of SAM.
[0020] FIG. 3 are representations of Western blots showing that the
complex formation between DNA MTase inhibitors and DNA MTase enzyme
is completed within 30 minutes.
[0021] FIG. 4 are representations of Western blots, panel A shows
results of complex formation studies in which nuclear extracts were
incubated with labeled oligonucleotide substrate (C) or inhibitor
(I or U), followed by addition of a 100-fold excess of unlabeled
substrate or inhibitor. Panel B shows results of complex formation
studies in which nuclear extracts were incubated with 50 .mu.M
unlabeled oligonucleotide substrate (C), inhibitor (I or U), or
with natural hemimethylated DNA substrate (HM) followed by addition
of 0.5 .mu.M labeled substrate or inhibitor.
[0022] FIG. 5 is a representation of a blot showing time-dependent
cellular uptake of a preferred embodiment of DNA MTase enzyme
inhibitors according to the invention.
[0023] FIG. 6 is a diagrammatic representation showing
dose-dependent inhibition of soft agar colony formation by Y1 cells
treated with antisense oligonucleotides complementary to DNA MTase
coding sequence.
[0024] FIG. 7 are diagrammatic representations showing reduction in
tumor size in tumor bearing mice treated with antisense
oligonucleotides complementary to DNA MTase coding sequence.
[0025] FIG. 8 are representations of blots showing the
intracellular localization in the nucleus of hemimethylated-hairpin
substrate of DNA Mtase 1 hour post treatment (Panel A), 4 hours
post treatment (Panel B), and 24 hours post treatment (Panel
C).
[0026] FIG. 9 is a diagrammatic representation showing inhibition
of DNA Mrase activity in cells treated with the hemimethylated test
inhibitor having the sequence of SEQ. ID. NO. 13 at a concentration
of 100 nM.
[0027] FIG. 10 is a diagrammatic representation showing an overall
reduction in the percentage of non-methylated CG dinucleotides in
DNA MTase inhibitor-treated cells as compared to untreated
cells.
[0028] FIG. 11 is a diagrammatic representation showing a
dose-dependent reduction in growth on soft agar observed following
treatment with DNA Mase enzyme inhibitors according to the
invention.
[0029] FIG. 12 is a diagrammatic representation showing a
dose-dependent reduction in cell number following treatment with
DNA MTase enzyme inhibitors according to the invention.
[0030] FIG. 13 is a diagrammatic representation of a gel shift
assay showing the binding of human DNA MTase to the MTase enzyme
inhibitors according to the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0031] The invention relates to modulation of gene expression. In
particular, the invention relates to modulation of gene expression
of the gene encoding DNA methyltransferase, and to modulation of
gene expression that is regulated by the enzyme DNA
methyltransferase. The patents and publications identified in this
specification are within the knowledge of those skilled in this
field and are hereby incorporated by reference in their
entirety.
[0032] The invention provides novel inhibitors of DNA MTase enzyme
and methods for using such inhibitors as analytical and diagnostic
tools, as potentiators of transgenic plant and animal studies and
gene therapy approaches, and as potential therapeutic agents.
[0033] In a first aspect, the invention provides novel hairpin
oligonucleotide inhibitors of DNA methyltransferase (DNA MTase)
enzyme.
[0034] As used herein, "DNA methyltransferase" is a protein which
is capable of methylating a particular DNA sequence.
[0035] In a preferred embodiment, the normal substrate for DNA
MTase is a hemimethylated double stranded DNA molecule having a CG
dinucleotide opposite a 5-methyl CG dinucleotide, e.g., in a
hairpin forming oligonucleotide, and methylation occurs at the
5-position of the cytosine base in the CG dinucleotide. Most
preferably, the DNA methyltransferase is mammalian DNA
methyltransferase or M.SssI DNA methyltransferase.
[0036] The present inventors have discovered that substitution of
the CG dinucleotide with a phosphorothioate IG, UG,
5-bromocytosineG, 5-fluorocytosineG, abasicG, or CG dinucleotide in
a hairpin forming oligonucleotide results in a powerful
mechanism-based inhibitor of DNA MTase. Thus, inhibitors according
to this aspect of the invention have the general structure: 3
[0037] wherein each N is independently any nucleotide, n is a
number from 0-20, C is 5-methylcytidine, G is guanidine, y is a
number from 0-20, L is a linker, each D is a nucleotide that is
complementary to an N such that Watson-Crick base pairing takes
place between that D and the N such that the N.sub.n--C--G--N.sub.y
and the D.sub.n--G--B--D.sub.y form a double helix, B is cytosine,
inosine, uridine, 5-bromocytosine, abasic deoxyribose, or
5-fluorocytosine, dotted lines between nucleotides represent
hydrogen bonding between the nucleotides, the linkage between G and
B is a phosphorothioate or phosphorodithioate linkage and the total
number of nucleotides ranges from about 10 to about 50. In one
particularly preferred embodiment, L is a nucleoside or an
oligonucleotide region having from 2 to about 10 nucleotides.
Preferably, the indicated CG and GB and about 2 flanking
nucleotides on either side are deoxyribonucleosides. DNA MTase
inhibitors according to this aspect of the invention bind DNA MTase
enzyme avidly in a noncovalent manner and inhibit DNA MTase in an
S-adenosylmethionine (SAM)-independent manner.
[0038] Examples of certain preferred DNA MTase inhibitors according
to this aspect of the invention include those having the following
nucleotide sequences (hydrogen bonding not shown): 4
[0039] wherein C is cytidine, T is thymidine, A is adenosine, G is
guanine, m is a methyl group at the 5-position of cytosine, B is
cytosine, inosine, uridine, 5-bromocytidine, or 5-fluorouridine, X
is any base and F is 5-fluorocytosine, and wherein the methyl
acceptor site is a phosphorothioate or phosphorodithioate
dinucleoside. As the methyl group is not actually transferred,
"methyl acceptor site" means the dinucleoside which is substituted
for the natural CG dinucleoside which would have been the methyl
acceptor site.
[0040] From the foregoing, those skilled in the art will recognize
that the overall sequence of the oligonucleotide inhibitor of DNA
MEase is not critical, so long as it is capable of forming a
hairpin oligonucleotide in which one strand has a 5-methylCG
dinucleotide and the other strand has, opposite the 5-methylCG
dinucleotide, an IG, UG, 5-bromocytosineG, abasicG,
5-fluorocytosineG or CG dinucleoside phosphorothioate or
phosphorodithioate, preferably with both nucleosides being
deoxyribonucleosides. In a preferred embodiment, seven or more
consecutive nucleosides, including the methyl acceptor site, are
phosphorothioate linked. Preferably, when two or more CG
dinucleosides are present, they are spaced apart, rather than being
in tandem. Where two or more CG dinucleosides are present, it is
preferred that at least the CG dinucleoside furthest from the
linker be a phosphorothioate or phosphorodithioate dinucleoside. In
certain embodiments, two or more CG dinucleosides, or at least one
CG and one TG nucleoside, are preferred. In such embodiments, the
CG furthest from the linker is generally opposite the methyl
acceptor site. It is preferred that at least one nucleoside on the
strand which does not contain the methyl acceptor site be
methylated, most preferably being a methylcytosine or a thymidine
nucleoside. Where such nucleoside is opposite the G of the methyl
acceptor site, methylcytosine is particularly preferred. Preferably
the hairpin structure contains at least three base pairs, more
preferably at least 6, and most preferably at least 12.
[0041] For purposes of the invention, the term "oligonucleotide"
includes polymers of two or more deoxyribonucleotide,
ribonucleotide, or 2'-O-substituted ribonucleotide monomers, or any
combination thereof. Such monomers may be coupled to each other by
any of the numerous known internucleoside linkages. In certain
preferred embodiments, these internucleoside linkages may be
phosphodiester, phosphotriester, phosphorothioate, or
phosphoramidate linkages, or combinations thereof. In a
particularly preferred embodiment the monomers are coupled by one
or more phosphorothioate linkages.
[0042] The term oligonucleotide also encompasses such polymers
having chemically modified bases or sugars and/or having additional
substituents, including without limitation lipophilic groups,
intercalating agents, diamines and adamantane. For purposes of the
invention the term "2'-O-substituted" means substitution of the 2'
position of the pentose moiety with an --O--lower alkyl group
containing 1-6 saturated or unsaturated carbon atoms, or with an
--O--aryl or allyl group having 2-6 carbon atoms, wherein such
alkyl, aryl or allyl group may be unsubstituted or may be
substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano,
nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino
groups; or such 2' substitution may be with a hydroxy group (to
produce a ribonucleoside), an amino or a halo group, but not with a
2'-H group.
[0043] Inhibitors according to the invention may conveniently be
synthesized on a suitable solid support using well known chemical
approaches, including H-phosphonate chemistry, phosphoramidite
chemistry, or a combination of H-phosphonate chemistry and
phosphoramidite chemistry (i.e., H-phosphonate chemistry for some
cycles and phosphoramidite chemistry for other cycles). Suitable
solid supports include any of the standard solid supports used for
solid phase oligonucleotide synthesis, such as controlled-pore
glass (CPG). (See, e.g., Pon, Methods in Molec. Biol. 20:465
(1993)).
[0044] DNA MTase inhibitors according to the invention are useful
for a variety of purposes. For example, they can be used as
"probes") of the physiological function of DNA MTase by being used
to inhibit the activity of DNA MTase in an experimental cell
culture or animal system and to evaluate the effect of inhibiting
such DNA MTase activity. This is accomplished by administering to a
cell or an animal a DNA MTase inhibitor according to the invention
and observing any phenotypic effects. In this use, DNA MTase
inhibitors according to the invention are preferable to traditional
"gene knockout" approaches because they are easier to use and can
be used to inhibit DNA MTase activity at selected stages of
development or differentiation. Thus, DNA MTase inhibitors
according to the invention can serve as probes to test the role of
DNA methylation in various stages of development.
[0045] DNA MTase inhibitors according to the invention are useful
as diagnostic probes for whether a cell sample is cancerous, as
described in detail elsewhere in this specification. In addition,
DNA MTase inhibitors according to the invention are useful for in
vivo imaging of cancer cells. Since cancer cells have elevated
levels of DNA MTase and normal cells do not, DNA MTase inhibitors
according to the invention will form stable complexes with DNA
MTase in cancer cells, but not in normal cells. Thus, appropriate
labeling of DNA MTase inhibitors with an imaging agent, e.g.,
technecium, will result in localization of the label at the site of
the cancer cells. This effect may be enhanced by using DNA MTase
inhibitors which are unstable (e.g., oligonucleotide
phosphodiesters) or rapidly cleared (e.g., oligonucleotide
methylphosphonates) in the absence of complex formation, thus
reducing background noise.
[0046] Finally, DNA MTase inhibitors according to the invention are
useful in therapeutic approaches to cancer and other diseases
involving suppression of gene expression. The anti-cancer utility
of DNA MTase inhibitors according to the invention is described in
detail elsewhere in this specification. In addition, DNA MTase
inhibitors according to the invention may be used to activate
silenced genes to provide a missing gene function and thus
ameliorate disease symptoms. For example, the diseases beta
thalassemia and sickle cell anemia are caused by aberrant
expression of the adult beta globin gene. Most individuals
suffering from these diseases have normal copies of the fetal gene
for beta globin. However, the fetal gene is hypermethylated and is
silent. Activation of the fetal globin gene could provide the
needed globin function, thus ameliorating the disease symptoms.
[0047] For therapeutic use, DNA MTase inhibitors according to the
invention may optionally be formulated with any of the well known
pharmaceutically acceptable carriers or diluents. This formulation
may further contain one or more additional DNA MTase inhibitors
according to the invention. Alternatively, this formulation may
contain one or more anti-DNA MTase antisense oligonucleotide or it
may contain any other pharmacologically active agent.
[0048] In a second aspect, the invention provides inhibitors of DNA
MTase enzyme which also inhibit the expression of the DNA MTase
gene. Inhibitors according to this aspect of the invention have the
general structure: 5
[0049] wherein the substituents are the same as for inhibitors
according to the first aspect of the invention, except that X is an
antisense oligonucleotide of from about 10 to about 50 nucleotides
in length, which is complementary to a portion of an RNA encoding
DNA MTase enzyme, and L can optionally be X. Particularly preferred
embodiments have the antisense oligonucleotide coupled at one or
both ends to one or more of the inhibitors selected from the group
consisting of SEQ. ID. NOS. 1-41.
[0050] In a third aspect, the invention provides a diagnostic
method for determining whether a particular sample of cells is
cancerous. The method according to this aspect of the invention
comprises preparing an extract from the cells in the cell sample,
adding labelled inhibitor according to the invention, measuring the
extent of formation of a complex between the labeled inhibitor and
DNA MTase enzyme, normalizing the extent of such complex formation
to the number of cells represented in the sample to obtain a
normalized complex formation value, and comparing the normalized
complex formation value to a normalized complex formation value for
non-cancerous and/or cancerous cell samples. In a preferred
embodiment, the extract is a nuclear extract. Because cancer cells
express DNA MTase at much higher levels than do non-cancerous
cells, the comparison of the normalized complex formation values is
diagnostic for whether the cell sample is cancerous.
[0051] In the diagnostic method according to this aspect of the
invention, the extent of complex formation may be carried out in a
variety of ways. For example, radiolabeled inhibitor may be used
and the extent of incorporation of the inhibitor into a complex of
appropriate size (e.g., 190 kDa for a 27-mer inhibitor) can be
determined. Alternatively, anti-DNA MTase antisera can be employed
to determine the quantity of complex of appropriate size which is
present. In another embodiment, antibodies or other binding
partners can be prepared which recognize only the complex formed
between the inhibitor and DNA MTase, and thus can be used to
measure it formation. Normalizing the extent of complex formation
to the number of cells can similarly be carried out in a variety of
ways. For example, the number of cells in the test sample, as well
as in the non-cancerous and cancerous control samples, can be
counted prior to extract formation. Alternatively, the total amount
of protein in each of the extracts can be determined using standard
procedures. The normalized complex formation value can then be
determined by dividing the extent of complex formation by the
number of cells in the sample or the amount of protein in the
extract.
[0052] In a fourth aspect, the invention provides methods for
inhibiting tumorigenesis comprising administering to an animal,
including a human, inhibitors according to the invention. In the
method according to this aspect of the invention a therapeutically
effective amount of a DNA MTase inhibitor according to the
invention is administered for a therapeutically effective period of
time to an animal, including a human, which has cancer cells
present in its body. Preferably, such administration should be
parenteral, oral, sublingual, transdermal, topical, intranasal or
intrarectal. Administration of the therapeutic compositions can be
carried out using known procedures at dosages and for periods of
time effective to reduce symptoms or surrogate markers of the
cancer. When administered systemically, the therapeutic composition
is preferably administered at a sufficient dosage to attain a blood
level of DNA MTase inhibitor from about 0.01 micromolar to about 10
micromolar. For localized administration, much lower concentrations
than this may be effective, and much higher concentrations may be
tolerated. Preferably, a total dosage of DNA methyltransferase
inhibitor will range from about 0.1 mg oligonucleotide per patient
per day to about 200 mg oligonucleotide per kg body weight per day.
It may desirable to administer simultaneously, or sequentially a
therapeutically effective amount of one or more of the therapeutic
compositions of the invention to an individual as a single
treatment episode.
[0053] The following examples are intended to further illustrate
certain preferred embodiments of the invention and are not limiting
in nature.
EXAMPLE 1
[0054] Inhibition of DNA MTase Activity in Nuclear Extracts
Prepared from Human or Murine Cells
[0055] Nuclear extracts were prepared from 1.times.10.sup.8 mid-log
phase human H446 cells, human A549 cells or mouse Y1 cells. The
cells were harvested and washed twice with phosphate buffered
saline (PBS), then the cell pellet was resuspended in 0.5 ml Buffer
A (10 mM Tris pH 8.0, 1.5 mM MgCl.sub.2, 5 mM KCl.sub.2, 0.5 mM
DTT, 0.5 mM PMSF and 0.5% Nonidet P40) to separate the nuclei from
other cell components. The nuclei were pelleted by centrifugation
in an eppendorf microfuge at 2,000 rpm for 15 min at 4.degree. C.
The nuclei were washed once in Buffer A and repelletted, then
resuspended in 0.5 ml Buffer B (20 mM Tris pH 8.0, 0.25% glycerol,
1.5 mM MgCl.sub.2, 0.5 mM PMSF, 0.2 mM EDTA 0.5 mM DTT and 0.4 mM
NaCl). The resuspended nuclei were incubated on ice for 15 minutes
then spun at 15,000 rpm to pellet nuclear debris. The nuclear
extract in the supernatant was separated from the pellet and used
for assays for DNA MTase activity. For each assay, carried out in
triplicate, 3 micrograms of nuclear extract was used in a reaction
mixture containing 0.1 micrograms of a synthetic 33-base pair
hemimethylated DNA molecule substrate with 0.5 .mu.Ci
S-[methyl-.sup.3H] adenosyl-L-methionine (78.9 Ci/mmol) as the
methyl donor in a buffer containing 20 mM Tris HCl (pH 7.4), 10 mM
EDTA, 25% glycerol, 0.2 mM PMSF, and 20 mM 2-mercaptoethanol. The
reaction mixture was incubated for 1 hour at 37.degree. C. to
measure the initial rate of the DNA MTase. The reaction was stopped
by adding 10% TCA to precipitate the DNA, then the samples were
incubated at 40.degree. C. for 1 hour and the TCA precipitates were
washed through GFC filters (Fischer). Controls were DNA incubated
in the reaction mixture in the absence of nuclear extract, and
nuclear extract incubated in the reaction mixture in the absence of
DNA. The filters were laid in scintillation vials containing 5 ml
scintillation cocktail and tritiated methyl groups incorporated
into the DNA were counted in a beta scintillation counter. To
measure inhibition of DNA MTase activity by different inhibitors,
parallel reactions were carried out in which the inhibitors were
added to the reaction mixtures in increasing concentrations ranging
from 1 to 1000 nM. The control inhibitors had the same nucleotide
sequence as the test inhibitors, except that the control inhibitor
had an o-methyl modified ribose, or was a scrambled
oligonucleotide, whereas the test inhibitors had either an IG or UG
dinucleotide, or a bromocytosine G, or a 5-fluorocytosine G
dinucleotide or a cytosine opposite the 5-methylCG dinucleotide.
The EC.sub.50 was calculated as the concentration of inhibitor
required to inhibit 50% of the DNA MTase activity present in the
nuclear extract.
[0056] Test inhibitors showed an EC.sub.50 of less than 1 .mu.M
with some embodiments showing an EC.sub.50 of as low as 30 nM. See
FIG. 1. The control inhibitor could not produce an EC.sub.50 at any
concentration tested (up to 1 .mu.M). Representative data using the
test inhibitors of the invention are shown in table 1.
1 TABLE 1 EC.sub.50 SEQ. ID. NO. 30 nM 1 30 nM 2 350 nM 4 30 nM 13
50 nM 14 50 nM 15 350 nM 16 350 nM 17 450 nM 18 450 nM 19 500 nM 20
500 nM 21 500 nM 22 230 nM 23 230 nM 24 300 nM 25 300 nM 26 300 nM
27 300 nM 28 50 nM 29 500 nM 30 700 nM 31 600 nM 32 600 nM 33 650
nM 34 35 nM 35 400 nM 36 500 nM 37 500 nM 38 500 nM 39 230 nM 40
230 nM 41
[0057] These results demonstrate that substitution of an IG or UG
or CG dinucleotide, or a 5-bromocytosine G, or a 5-fluorocytosine G
dinucleotide opposite a 5-methylCG dinucleotide in a synthetic
hairpin forming oligonucleotide results in an effective inhibitor
of DNA MTase activity.
[0058] The results shown in FIG. 9 demonstrate that the
hemimethylated test inhibitor having the sequence of SEQ. ID. NO.
13 at a concentration of 100 nM inhibited DNA MTase activity as
compared to the control nonmethylated hairpin.
EXAMPLE 2
[0059] Complex Formation Between DNA MTase Inhibitors and DNA MTase
Enzyme
[0060] To measure the rate of complex formation between different
DNA MTase inhibitors and DNA MTase enzyme, different inhibitors
(each at 4 micromolar concentration) were labeled using
polynucleotide kinase and gamma .sup.32P-ATP (300 mCi/mmol, 50
.mu.Ci) (New England Biolabs, Beverly, Mass.) as recommended by the
manufacturer. Labeled oligonucleotide was separated from
nonincorporated radioactivity by passing through a G-50 Sephadex
spin column (Pharmacia, Uppsala, Sweden). Labeled inhibitors (500
nM) were incubated with 5 micrograms nuclear extract prepared as
described in Example 1. The incubation, in the same buffer used for
the DNA MTase activity assay, was at 370.degree. C. for 30 minutes.
To determine whether complex formation was dependent on the
cofactor SAM, the reaction was carried out both in the presence and
the absence of SAM). Then, loading dye (0.3 M Tris-HCl pH 8.8, 0.2%
SDS, 10% glycerol, 28 mM 2-mercaptoethanol and 24 .mu.g/ml
bromophenol blue) was added and the sample was separated on a 5%
SDS-polyacrylamide gel (SDS-PAGE) with a 4% stacking gel according
to standard procedures. Following SDS-PAGE separation, the gel was
exposed to autoradiography for visualization of a complex migrating
at 190 kDa. Alternatively, the gel was electrotransferred onto a
PVDF membrane (Amersham Life Sciences, Buckinghamshire, England)
using a BioRad (Hercules, Calif.) electrotransfer apparatus at 250
milliamperes for 2.5 hours in electrotransfer buffer (3.03 g/l Tris
base, 14.4 g/l glycine, 1 g/l SDS, pH 8.3) for Western blotting
with a DNA MTase-specific antisera. The membrane was blocked for 1
hour in a buffer containing 5 mM Tris base, 200 mM NaCl, 0.5%
Tween-20 and 5% dry milk. Rabbit antisera was raised according to
standard procedures against a peptide sequence found in the
catalytic domain of human and murine DNA MTase. The antisera was
added to the membrane at a 1:200 dilution and incubated for 1 hour.
The membrane was washed with the blocking buffer, then reacted with
a 1:5000 dilution of goat anti-rabbit secondary antibody (Amersham)
for an additional hour. The membrane was then washed for 10 minutes
in blocking buffer, three times, and bands reacting with anti-DNA
MTase antibody were visualized using an ECL detection kit
(Amersham).
[0061] Typical results are shown in FIGS. 2 and 3. The results
shown in FIG. 2 demonstrate that a 190 kDa complex is detected by
both autoradiography and Western blotting, strongly indicating that
the 190 kDa complex is formed between the DNA MTase inhibitors and
DNA MTase enzyme. These results further demonstrate that such
complex formation is independent of the cofactor SAM. The results
shown in FIG. 3 demonstrate that the complex formation is complete
within 30 minutes, thus suggesting that such complex formation
provides an assay for the level of DNA MTase in different cell
samples.
[0062] In addition, nuclear extracts prepared as described in
Example 1, were incubated with labeled inhibitors to perform gel
shift assays according to standard protocols (see e.g., Molecular
Cloning, 2d Edition, Cold Spring Harbor Laboratory Press (1989)).
The results shown in FIG. 13, further demonstrate the binding of
human DNA methyltransferase to the inhibitors of the invention.
EXAMPLE 3
[0063] Stability of Noncovalent Complex Formation Between DNA MTase
Inhibitors and DNA MTase Enzyme
[0064] To determine the stability of the complex between DNA MTase
inhibitors and DNA MTase enzyme, relative to the stability of the
complex between normal substrate and DNA MTase enzyme, binding
competition assays were carried out as follows. Complex formation
was carried out as described in Example 2, except that the labeled
substrate or inhibitor was allowed to form a complex with the DNA
MTase, followed by addition of a 100-fold excess of unlabeled
substrate or inhibitor. The substrate was a hairpin forming
oligonucleotide having a 5-methylCG dinucleotide on one strand,
opposite a CG dinucleotide on the other strand. The inhibitors were
identical, except that they had an IG or UG dinucleotide on the
other strand. The results are shown in FIG. 4, panel A. Where
complex formation was originally carried out using the substrate
oligonucleotide, no radiolabeled complex was detected, suggesting
that the complex is labile. However, where complex formation was
originally carried out using either the IG or the UG inhibitor, the
excess unlabeled substrate or inhibitor was unable to displace the
radiolabel from the complex, indicating that the DNA MTase
inhibitor-DNA MTase enzyme complex is very stable, with a slow off
rate.
[0065] Alternatively, when 50 .mu.M unlabeled substrate or
inhibitor was pre-incubated with the nuclear extract, subsequent
incubation with 0.5 .mu.M radiolabeled substrate or inhibitor could
not displace the unlabeled substrate or inhibitor from the complex.
However, the labeled inhibitor could displace unlabeled
hemi-methylated DNA, the natural substrate for DNA MTase, from such
a pre-formed complex. These results demonstrate that binding of the
inhibitors to DNA MTase enzyme is specific and saturable, and that
such inhibitors are efficacious competitors of the natural
substrate of DNA MTase (FIG. 4, panel B).
EXAMPLE 4
[0066] DNA MTase Inhibitor Accumulation and Formation of Complexes
with DNA MTase Enzyme in Cells
[0067] DNA MTase inhibitors were labeled with .sup.32P as described
in Example 2. 300,000 Y1 cells were plated per well in a six well
tissue culture plate. Labeled inhibitors were added to a final
concentration of 1 micromolar. Cells were harvested at different
time points by trypsinization and washed extensively .with PBS to
remove nonincorporated compounds. The cell pellet was resuspended
in 20 pl buffer RIPA (0.5% deoxycholic acid, 0.1% SDS, 1% NP-40, in
PBS). The homogenate was incubated at 4.degree. C. for 30 minutes,
then spun in a microfuge at maximum speed for 30 minutes, after
which the supernatant was transferred to a new tube. Two .mu.l of
supernatant were extracted with phenol-chloroform and loaded onto a
20% polyacrylamide-urea gel. Visualization was by autoradiography.
The results demonstrated that the DNA MTase inhibitors were taken
up by the cells in a time-dependent manner (FIG. 5). Ten
microliters of the supernatant are loaded directly on 5% SDS-PAGE
and visualized by autoradiography to detect complex formation. It
is expected that the 190 kDa complex between the 27-mer DNA MTase
inhibitor and DNA MTase enzyme will be observed.
EXAMPLE 5
[0068] Intracellular Localization of DNA MTase Inhibitors
[0069] 100 nM of oligonucleotide having the sequence shown as SEQ.
ID. NO. 22 were labeled with the mixed isomeric
N-hydroxysuccinimide esters of 5(G)-carboxyfluorescein (Molecular
Probes, Eugene, Oreg.) as described by Sinha and Striepeke in
Oligonucleotides and Analogues: A Practical Approach
(1991)(Eckstein, Ed.) Oxford University Press, NY) pp 185-210. In a
typical reaction, 28 nmol of oligonucleotide was dissolved in 180 1
of 0.4 M NaHCO.sub.3/Na.sub.2CO.sub.3 pH 9.0, 1 M
N-dimethylformamide/wat- er (3:2:1 v/v/v). This solution was
diluted with an equal volume of water, and 1.5 mg of active ester
of the fluorophore was added. The mixture was kept at room
temperature in the dark for 17 h with gentle shaking and then
diluted to 6 ml with water. Most of the excess dye was removed by
extraction of the aqueous solution with n- butanol followed by
ethanol precipitation according to standard methods. Human lung
carcinoma A549 cells were grown to 10-5 and incubated with the
labeled inhibitor in the presence of 10 .mu.g/me lipofectin. The
subcellular localization of the labeled inhibitor at different time
points was determined by fluorescent microscopy. As shown in FIG.
8, a diffuse pattern of distribution of the test inhibitor in the
cytosol and in the nuclei was observed 1 hour post treatment. At 4
hours post treatment almost all the test inhibitor was localized in
the nucleus the site of action of DNA MTase. At 24 hours, the test
inhibitor was mainly in the nucleus distributed in a punctate
manner which is similar to the pattern of localization of DNA
MTase. This experiment demonstrates that the DNA inhibitor is
localized in the site of action of DNA MTase.
EXAMPLE 6
[0070] Analysis of Cellular DNA Methylation in Cells Treated with
DNA MTase Inhibitors
[0071] Nuclear extracts were prepared from untreated cells and from
DNA MTase inhibitor-treated cells (1 .mu.M inhibitor having SEQ ID
NO: 13) according to the methodology described in Example 1. The
DNA pellet was resuspended in 0.5 ml DNA extraction buffer (0.15 M
NaCl, 1% SDS, 20 mM Tris-HCl pH 8.0, 5 mM EDTA), then 100 .mu.g
protease K was added and the suspension was incubated at 50.degree.
C. for 16 hours. The DNA was extracted in phenol-chloroform by
adding 0.25 ml phenol and 0.25 ml chloroform. The suspension was
mixed and the organic and aqueous phases were separated by
centrifugation in a microfuge for 10 minutes at 15,000 rpm. One ml
absolute ethanol was added to the aqueous phase and the DNA was
precipitated by centrifugation in a microfuge for 15 minutes at
15,000 rpm. The DNA pellet was washed in 70% ethanol and repelleted
by centrifugation. The DNA was resuspended in 100 .mu.l 20 mM
Tris-HCl pH 8.0, 1 mM EDTA.
[0072] Two .mu.g DNA were incubated at 37.degree. C. for 15 minutes
with 0.1 unit of DNAase, 2.5 .mu.l .sup.32P-alpha-dGTP (3000
Ci/mmol, Amersham) and then 2 units Kornberg DNA polymerase
(Boehringer Mannheim, Mannheim, Germany) were added and the
reaction mixture was incubated for an additional 25 minutes at
30.degree. C. Fifty .mu.l water was then added and nonincorporated
radioactivity was removed by spinning through a Microspin S-300 HR
column (Pharmacia). Labelled DNA (20 .mu.l) was digested with 70
.mu.g micrococcal nuclease (Pharmacia) in the manufacturer's
recommended buffer for 10 hours at 37.degree. C. Equal amounts of
radioactivity were loaded onto TLC phosphocellulose plates (Merck,
Darmstadt, Germany) and the 3' mononucleotides were separated by
chromatography in one direction, in 66:33:1 isobutyric
acid/H.sub.2O/NH.sub.4OH. The chromatograms were exposed to XAR
film (Eastman Kodak, Rochester, N.Y.) and the autoradiograms were
scanned by laser densitometry (Scanalytics, CSPI, Billerica,
Mass.). Spots corresponding to cytosine and 5-methylcytosine were
quantified and the percentage of non-methylated CG dinucleotides
was determined. The results shown in FIG. 10 demonstrate an overall
reduction in the percentage of non-methylated CG dinucleotides in
DNA MTase inhibitor-treated cells, relative to untreated cells.
[0073] To asses demethylation of specific genes, a procedure is
carried out as generally described in J. Biol. Chem.
270:12690-12696 (1995). Briefly, the genomic DNA (10 .mu.g) is
extracted and subjected to digestion by 25 units HindIII, followed
by digestion by either 25 units MspI (CG methylation insensitive)
or 25 units HpaII (CG methylation sensitive) for 8 hours at
37.degree. C. The digested DNA is separated on a 1.5% agarose gel
and subjected to Southern blotting and hybridization with specific
probes. The results are expected to show that genes which are
ordinarily heavily methylated in the test cells become
undermethylated, whereas the methylation levels for genes which are
not ordinarily heavily methylated in the test cells are not
significantly affected.
EXAMPLE 7
[0074] Inhibition of Tumorigenesis By Inhibitors of DNA MTase A549
cells were plated on a 6 well plate at a density of 80,000
cells/well. DNA MTase enzyme inhibitors (from about 10 to about
1000 nanomolar) or antisense oligonucleotide phosphorothioates
complementary to the DNA MTase coding sequence (about 0.5 to 20
micromolar) were added to the cells. The cells were similarly
treated daily for 3 days. Then, the cells were harvested and 3,000
live cells were plated in soft agar, as described in Freedman and
Shin, Cell 3:355-359 (1974). Two weeks after plating, the number of
colonies formed in soft agar were scored by visual examination. In
the case of antisense oligonucleotides, a dose-dependent reduction
in the number of colonies was observed (FIG. 6). The results shown
in FIG. 11 demonstrate a dose-dependent reduction in growth on soft
agar observed following 3 days treatment with DNA MTase enzyme
inhibitors. The results shown in FIG. 12 demonstrate a
dose-dependent reduction cell number treatment with DNA MTase
enzyme inhibitors.
[0075] Alternatively, 6 to 8 week old LAF-1 mice (Jackson Labs, Bar
Harbor, Me.) are injected subcutaneously in the flank area with
2.times.10.sup.6 Y1 cells. Three days later, the mice are injected
with 1-5 mg/kg antisense oligonucleotide phosphorothioates
complementary to DNA MTase coding sequence, or with 5 mg/kg DNA
MTase enzyme inhibitor. This dosing is repeated every two days.
After one month, the mice are sacrificed and the tumor size is
determined. In the case of the antisense oligonucleotides,
significant reduction in tumor size was observed, relative to
controls treated with a randomized or a reverse antisense sequence
(FIG. 7). Similar results are expected for the DNA MTase enzyme
inhibitors.
* * * * *