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`
`

`

`[Epigenetics 1:1, 7-13, January/February/March 2006]; ©2006 Landes Bioscience
`
`Review
`Reactivation of Epigenetically Silenced Genes by DNA Methyltransferase
`Inhibitors
`Basic Concepts and Clinical Applications
`
`Cora Mund
`Bodo Brueckner
`Frank Lyko*
`
`ABSTRACT
`Hypermethylation of tumor suppressor genes is one of the most consistent hallmarks of
`human cancers. This epigenetic alteration has been associated with gene silencing and
`thus represents an important pathway for generating loss-of-function mutations. In this
`review, we survey the available literature on systematic, genome-wide approaches aimed
`at the identification of epigenetically silenced loci. These studies uncovered a variety of
`diverse genes, but a common signature for epigenetic reactivation has not been identified.
`Nevertheless, DNA methyltransferase inhibitors have shown significant clinical benefits,
`mostly in the therapy of leukemias. Recent analyses revealed substantial drug-induced
`methylation changes that can now be used as endpoints for the further refinement of clinical
`treatment schedules. Further optimization of epigenetic cancer therapies should be feasible
`through the use of novel DNA methyltransferase inhibitors with improved specificity.
`Rational design of epigenetic inhibitors might provide the foundation for a broader use
`of these drugs in the treatment of cancer.
`
`Epigenetic mechanisms play a fundamental role in the interpretation of genetic infor-
`mation.1 Depending on its particular epigenetic modification pattern, a gene can be
`expressed or silenced. Epigenetic modifications thus represent an integral mechanism for
`the control of complex gene expression patterns. One prominent example for an epigenetic
`control mechanism is the covalent modification of histones.2 Histones can be modified at
`various amino acid residues by acetylation, phosphorylation, methylation and ubiquitina-
`tion3 and it has been suggested that particular combinations of these modifications
`constitute an “epigenetic code“ for the regulation of gene expression.2 Another prominent
`epigenetic modification is the methylation of cytosine residues in genomic DNA.4 About
`4% of the cytosines are usually methylated in mammalian genomic DNA5 and it has been
`shown that this methylation is essential for mouse development.6 In addition, it has also
`been shown that DNA methylation plays an essential role in several epigenetic phenomena,
`including genomic imprinting,7 X-chromosome inactivation,8 and retroelement silencing.9
`CpG dinucleotides represent the consensus target sequences of DNA methylation in
`differentiated mammalian cells, but only a relatively small fraction of these sequences
`becomes methylated.10 CpG dinucleotides can be clustered in CpG islands11,12 which are
`often associated with promoter regions and remain unmethylated for most genes. The
`signals that determine whether a particular CpG-containing sequence becomes methylated
`have not been determined yet, but it has been suggested that protein-protein interactions
`between DNA methyltransferases and chromatin-associated proteins might play an important
`role.13 As a consequence, DNA methylation is not evenly distributed over the genome.
`While the majority of repetitive elements are usually heavily methylated, gene-specific
`methylation appears to be rather restricted.
`Genomic DNA methylation patterns can be drastically altered in human tumors.14
`Due to the selective forces driving carcinogenesis, tumor cells are characterized by specific
`epigenetic changes that promote uncontrolled cellular proliferation.15 This explains the
`remarkable consistency of gene hypermethylation across all forms of human cancers.16
`When hypermethylation affects the CpG islands of genes it can trigger their stable repres-
`sion and thus has consequences that are functionally equivalent to genetic mutations.17
`The acquisition of such an “epimutation“ can therefore make an important contribution
`to cellular transformation. Over the past few years, the number of known epimutations
`has vastly increased. Prominent examples for genes “hit“ by hypermethylation-induced
`silencing include the cell-cycle regulators p15 and p16, the mismatch repair gene MLH1,
`as well as the apoptosis effector gene Apaf-1.18-20 These epimutations constitute a tumor-
`specific epigenetic program that is reflected by the typical characteristics of human cancer
`cells, the lack of cell cycle control and aggressive growth.
`
`Division of Epigenetics; Deutsches Krebsforschungszentrum; Im Neuenheimer Feld
`580; Heidelberg, Germany
`
`*Correspondence to: Frank Lyko, Ph.D.; Deutsches Krebsforschungszentrum; Im
`Neuenheimer Feld 580; 69120 Heidelberg, Germany; Tel.: +49.6221.423800;
`Fax: +49.6221.423802; Email: f.lyko@dkfz.de
`
`Received 11/04/05; Accepted 11/29/05
`
`Previously published as an Epigenetics E-publication:
`http://www.landesbioscience.com/journals/epigenetics/abstract.php?id=2375
`
`KEY WORDS
`
`DNA methylation, DNA methyltransferase
`inhibitors, epigenetic therapy
`
`©2006 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
`
`www.landesbioscience.com
`
`Epigenetics
`
`7
`
`

`

`DNA Methyltransferase Inhibitors
`
`E
`
`CR?
`I promoter
`
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`
`promoter
`
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`F
`
`promoter
`
`DNA methylation is catalyzed by DNA methyltransferases, a
`family of enzymes that comprises DNMT1, DNMT2, DNMT3A
`and DNMT3B in human cells.21 To analyze the functional role of
`individual DNA methyltransferases in hypermethylation-induced
`gene silencing, homologous recombination has been used to disrupt
`DNMT1 and DNMT3B in the colorectal cancer cell line
`HCT116.22,23 Lack of DNMT1 or DNMT3B had little effect on
`the DNA methylation pattern. However, a double knockout cell line
`lacking both enzyme activities showed a very strong reduction in
`5-methylcytosine content.23 The effects of DNA methyltransferase
`knockouts on genomic DNA methylation patterns have been ana-
`lyzed by two approaches:24 Differential methylation hybridization,
`which detects methylated regions in the genome through a CpG
`island microarray, and amplification of inter-methylated sites, which
`amplifies anonymous DNA sequences with a differential methylation
`pattern. It was demonstrated that cells lacking both DNMT1 and
`DNMT3B undergo a substantial loss of DNA methylation in the
`promoter region of tumor suppressor genes.24 This effect could not
`be seen in single DNMT knockout cell lines, and it was therefore
`concluded that different DNA methyltransferases cooperate in the
`epigenetic regulation of gene silencing.
`
`DNA METHYLTRANSFERASE INHIBITORS AS DEMETHYLATING
`AGENTS
`Based on the rationale that hypermethylation-induced gene
`silencing could be uncovered by gene demethylation and reactivation
`(Fig. 1), many laboratories have analyzed gene expression patterns in
`human cancer cells with experimentally reduced DNA methylation
`levels. To this end, DNA methyltransferase inhibitors,25 like 5-aza-
`cytidine, 5-aza-2'-deoxycytidine and, to a lesser extent, zebularine,
`have found widespread use. All three compounds need to be metab-
`olized and phosphorylated to deoxynucleotide triphosphates in
`order to become incorporated into DNA. DNA methyltransferases
`recognize the modified bases as natural substrate, but fail to resolve
`a covalent reaction intermediate,26 which results in the degradation
`of the covalently trapped enzymes. DNA demethylation is a direct
`consequence of enzyme trapping because of ongoing DNA replication
`in the presence of diminished DNA methyltransferase levels. After
`several rounds of DNA replication, this becomes detectable as a
`substantial decrease in the genomic methylation level.
`5-azacytidine is one of the best-known DNA methyltransferase
`inhibitors and has been used both in the laboratory and in the clinical
`practice for more than 20 years. The compound also represents the
`first established epigenetic drug, since it gained FDA approval for
`the treatment of myelodysplastic syndrome in May 2004. It was
`originally developed as a cytotoxic agent27 and its demethylating
`activity was only discovered through its ability to influence cellular
`differentiation.28 5-azacytidine is a ribose nucleoside and thus needs
`to be metabolized into a deoxyribonucleoside-triphosphate before it
`can be incorporated into DNA. Indeed, a major fraction of the drug
`becomes incorporated into RNA and thereby interferes with protein
`biosynthesis.29 This effect most likely plays a major role in the
`cytotoxicity of 5-azacytidine.
`5-aza-2'-deoxycytidine is the deoxyribose analogue of 5-azacytidine
`and a promising new drug for the treatment of myelodysplastic
`syndrome and other leukemias. This compound is a more potent
`hypomethylating agent, but it does not become incorporated into
`RNA. However, high doses of 5-aza-2'-deoxycytidine still induce
`substantial cytotoxicity, which is attributable to various side
`
`exon 1
`
`exon 1
`
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`Figure 1. Establishment and reversion of epigenetic mutations. In healthy cells,
`tumor suppressor genes are unmethylated and expressed at normal levels. In
`cancer cells, hypermethylation of the promoter (and/or exon 1) region leads
`to gene silencing. Treatment with DNA methyltransferase inhibitors (DNMT
`inhibitors) removes the majority of 5-methylcytosine (CH3), and can reactivate
`gene expression.
`
`effects.30 It was also shown that the differentiation inducing ability
`of decitabine in cultured fibroblasts had a narrow dose window,
`which provided a first indication that lower doses might induce
`demethylation with lower levels of overall cytotoxicity.28 Both 5-aza-
`cytidine and 5-aza-2'-deoxycytidine are unstable in aqueous solu-
`tions,31,32 which has limited their clinical application.
`The more recently discovered nucleoside inhibitor zebularine33 is
`a stable cytidine analog that has shown promising characteristics in
`several in vitro assays as well as in mouse models.34-36 Cancer cell
`lines that responded to zebularine by DNA demethylation showed a
`complete depletion of the DNMT1 methyltransferase and a partial
`depletion of DNMT3A and DNMT3B.36 Gene expression profiling
`revealed that zebularine specifically affected the gene expression
`patterns of human cancer cells, even if the number of genes upregu-
`lated by drug treatment was rather small (Table 1). Interestingly,
`zebularine was also shown to be active in mice when administered
`orally,34 which suggested novel treatment modalities for epigenetic
`therapies. However, a recent study has demonstrated very low oral
`bioavailability in monkeys37 and the clinical value of zebularine still
`remains to be determined.
`
`IDENTIFICATION OF NOVEL TUMOR SUPPRESSOR GENES
`BY EXPERIMENTAL DEMETHYLATION
`Because of the strong correlation between promoter hypermethy-
`lation and gene silencing, demethylating drugs have been repeatedly
`used for the identification of epigenetically silenced cancer genes.
`Transcriptional profiling in a variety of inhibitor-treated cancer cell
`lines revealed a relatively small number of genes that were significantly
`upor downregulated by demethylating drugs (Table. 1). Two studies
`suggested that genes in the interferon pathway might be induced by
`5-aza-2'-deoxcycytidine treatment,38,39 but, otherwise, no defined
`gene expression signatures have been identified. Based on the rationale
`that tumor suppressor genes become hypermethylated and silenced
`
`8
`
`Epigenetics
`
`2006; Vol. 1 Issue 1
`
`

`

`DNA Methyltransferase Inhibitors
`
`Table 1 Overview of gene expression changes induced by DNMT inhibitors
`
`Cell Line
`
`HT29
`
`T24
`
`LD419
`
`RKO
`
`KYSE30, KYSE410,
`KYSE520
`
`LNCaP
`
`DU145
`
`Drug
`
`No. of Genes
`Analyzed
`
`Genes
`Upreg. (%)
`
`Genes
`Downreg. (%)
`
`Reference
`
`5-aza-CdR
`
`5-aza-CdR
`
`5-aza-CdR
`
`5-aza-CdR
`
`5-aza-CdR
`
`5-aza-CdR
`
`5-aza-CdR
`
`4608
`
`6600
`
`6600
`
`10814
`
`12599
`
`1176
`
`1176
`
`0.4
`
`0.9
`
`0.5
`
`0.5
`
`1.0
`
`2.0
`
`1.6
`
`0.8
`
`n.d.
`
`0.03
`
`0.2
`
`n.d.
`
`n.d.
`
`2.0
`
`1.8
`
`0.6
`
`38
`
`39
`
`39
`
`41
`
`40
`
`77
`
`77
`
`43
`
`43
`
`36
`
`36
`
`44
`
`0.3
`
`0.05
`
`0.05
`
`0.4
`
`demethylation and were subsequently
`found to be hypermethylated in col-
`orectal cancers.41 More recently, it has
`been shown that epigenetic silencing of
`SFRP genes plays a functional role in
`the constitutive activation of the WNT
`signalling pathway, which further
`underscored the significance of these
`events for colorectal carcinogenesis.42
`To compare the effects of pharma-
`cological and genetic inactivation of
`DNA methyltransferases, transcriptional
`profiles of knockout cells were compared
`to those obtained from inhibitor-treated
`cells.43 Interestingly, the effects of
`5-aza-2'-deoxycytidine appeared fully
`established after 24 hours and more
`closely resembled those of histone
`deacetylase inhibitor (TSA) treatment
`than the effects of DNA methyltrans-
`ferase knockouts. These results cannot
`be explained by the covalent trapping
`of DNA methyltransferases, followed
`by passive demethylation of DNA.
`Rather, it seems likely that active
`demethylation is triggered by the
`pharmacological inhibitors through an
`upstream mechanism involved in the regulation of both DNA
`methylation and histone deacetylation. Another surprising finding
`was the substantial fraction of genes found to be downregulated after
`exposure to 5-aza-2'-deoxycytidine. In contrast to the prevailing
`view, this suggested that hypomethylation may also be associated
`with gene silencing. Consistently, demethylation of the APM2
`promoter region closely coincided with its transcriptional silencing.43
`The validity of the results obtained with cell lines was confirmed
`by a study that used primary cells from leukemia patients after treatment
`with 5-aza-2'-deoxycytidine ex vivo or in vivo.44 The transcriptional
`profiles obtained in these experiments were comparable to those
`obtained in cultured cell lines, which suggested that the molecular
`responses in cells and in patients might be similar. However, only
`half of the induced genes contained putative CpG islands in their 5'
`promoter region and only a fraction of those showed changes in their
`methylation patterns. This result provides a good starting point for
`a review of the complex data on drug-induced methylation changes
`in patient material.
`
`THE USE OF DEMETHYLATING DRUGS IN CLINICAL TRIALS
`Both 5-azacytidine and 5-aza-2'-deoxycytidine have been used in
`dozens of clinical trials for more than 20 years.45,46 In agreement
`with standard procedures, most early trials used the drugs at concen-
`trations that were close to the maximum tolerated dose. The clinical
`results were generally disappointing and included severe toxicities
`related to prolonged myelosuppression. More recently, treatment
`schedules have been adjusted to increase drug tolerance,47 which has
`allowed the completion of several effective low-dose trials and
`provided the foundation for an increased clinical interest in DNA
`methyltransferase inhibitors. A randomized, controlled phase III trial
`of low-dose 5-azacytidine vs. supportive care in myelodysplastic
`syndrome patients48 showed a significant clinical benefit in
`
`HCT116
`
`HCT116 (1KO)
`
`5-aza-CdR
`
`5-aza-CdR
`
`T24, HCT15, CFAPC-1
`
`zebularine
`
`LD98, LD419, T-1,
`CCD-1070K
`
`zebularine
`
`8000
`
`8000
`
`13300
`
`13300
`
`OCI-AML2
`
`5-aza-CdR
`
`22000
`
`5-aza-CdR: 5-aza-2’-deoxycytidine; n.d., not determined
`
`0.5
`
`0.1
`
`0.1
`
`0.4
`
`1.0
`
`0.8
`
`0.6
`
`5-azacytidine
`-
`- - supportive care
`
`Q)
`
`~ -..!.
`
`C
`Q)
`> Q)
`Cl
`.!=
`C ·ro
`E
`
`~ 0.4
`:0
`Cl)
`.0
`0
`C.
`
`0.0
`
`0
`
`6
`
`12
`
`36
`30
`24
`18
`time at risk [months]
`
`···-·-·· .................... ,_.,
`
`42
`
`48
`
`54
`
`~ 0.4 -0
`
`Figure 2. Clinical efficacy of DNA methyltransferase inhibitors in cancer
`patients. Survival of myelodysplastic syndrome patients treated with 5-azacy-
`tidine or supportive care, as determined by Kaplan-Meier analysis. Adapted
`from reference 48 with permission from the American Society of Clinical
`Oncology.
`
`in cancer cells, pharmacological inhibition of DNA methyltransferases
`was also used to uncover novel cancer-related genes. Transcriptional
`profiling of esophageal squamous cell carcinoma (ESCC) cell lines
`treated with 5-aza-2'-deoxycytidine identified ten putative tumor
`suppressor genes that were found to be methylated in primary tumor
`tissues.40 Three of these genes, CRIP-1, Apo D and NU were over-
`expressed in ESCC cells and found to strongly inhibit their ability to
`grow colonies.40 This provided an indication that all three genes
`might function as suppressors of tumor growth. In a similar study,
`the genes encoding secreted frizzled-related proteins (SFRPs) were
`identified through a genomic screen for genes upregulated by
`
`www.landesbioscience.com
`
`Epigenetics
`
`9
`
`

`

`DNA Methyltransferase Inhibitors
`
`diagnosis
`
`treatment
`
`monitoring
`
`I\
`sample O
`
`I\
`sample 2
`I\
`sample 3
`
`sample 1
`
`sample2
`
`sample 3
`
`sampleO
`
`drug-treated patients (Fig. 2). Similar results
`appear to have been obtained in a phase III
`clinical trial of 5-aza-2'-deoxycytidine.49 In
`addition, promising clinical responses have
`also been reported in phase I trials of low-
`dose 5-aza-2'-deoxycytidine in other leukemias,
`including AML and CML.50,51 These results
`suggest that the demethylation induced by
`azanucleoside drugs might be of general
`benefit for the reversion of epigenetic lesions
`in cancer patients.
`Drug-induced methylation changes have
`only recently been analyzed in the clinical
`setting. One of the first such studies focused
`on the methylation of the p15 tumor sup-
`pressor gene
`in the bone marrow of
`5-aza-2'-deoxycytidine-treated patients with
`myelodysplastic syndrome.52 The results
`suggested demethylation in nine out of 12
`patients and also provided evidence of p15
`reactivation by immunohistochemistry in
`four patients. It remains a possibility that
`these changes are at least partially attributable
`to the expansion of nonclonal cells in
`responding patients, but gene reactivation
`was also shown in morphologically dysplastic
`cells from patients that were not in complete
`remission. This indicated a significant poten-
`tial of the drug to activate gene expression in tumors and also
`suggested a direct involvement of DNA demethylation.52 However,
`there appeared to be no correlation between p15 demethylation and
`clinical response to decitabine in a larger phase I study of decitabine
`in MDS and AML patients.50 Overall similar results were also
`obtained in a phase I study of 5-azacytidine in Epstein-Barr
`virus-associated tumors.53 While these tumors showed substantial
`demethylation in the EBV promoter, reactivation of EBV expression
`could only be observed in one case and there appeared to be no
`detectable clinical response.
`Biological responses might be maximized by changes in the drug
`delivery schedule, and for this reason different schedules are currently
`being tested. In some of these clinical trials, the clinical responses are
`being analyzed in parallel with the changes in genomic DNA methy-
`lation patterns (Fig. 3). One such example is provided by a recent
`phase I study of continuous 5-aza-2'-deoxycytidine infusion in a
`small group of patients with refractory solid tumors.54 While the
`patient group was too small to assess the clinical benefit of the treat-
`ment schedule, all patients showed indications for demethylation in
`blood samples. This effect could be observed either by quantitative
`PCR-based methylation analysis of the MAGE-1 promoter or by
`HPLC analysis of genomic DNA. Importantly, it could also be
`shown that DNA methylation reverted to pretreatment levels within
`four weeks, which indicated that drug-induced demethylation is
`transient. These findings are in agreement with an analysis of drug-
`induced demethylation effects observed after 5-aza-2'-deoxycytidine
`treatment of MDS patients.55 Here, the analysis of global DNA
`methylation levels from serial bone marrow samples indicated strong
`demethylation after the fourth treatment cycle, while karyotype
`normalization had occurred after the second treatment cycle. This
`indicated an important role of drug-mediated cytotoxicity, followed
`by a substantial demethylation of nonclonal cells. Again, DNA
`
`Figure 3. Epigenetic cancer therapy. After diagnosis, cancer patients can be treated with DNA methyl-
`transferase and/or histone deacetylase inhibitors. Treatment response is being monitored at clinical
`and molecular levels by analysis of tumor and peripheral blood samples. Correlations between clinical
`and molecular parameters will be important for the optimization of clinical treatment schedules.
`
`methylation returned to pretreatment levels several weeks after the
`last treatment cycle, which confirmed the transient nature of the
`demethylating effect.
`The dynamics of drug-induced demethylation will be important
`for the design of combination trials that address the potential synergy
`between demethylating drugs and other chemotherapeutic agents.
`Experiments with mouse xenograft tumors have shown a strong
`chemosensitizing effect for 5-aza-2'-deoxycytidine when the drug
`was administered a few days before standard cytotoxic drugs.56 This
`effect is presumably due to the epigenetic reactivation of apoptosis
`effector and DNA repair genes. 5-aza-2'-deoxycytidine has also been
`shown to be effective in a clinical combination study with the tyrosine
`kinase inhibitor imatinib mesylate (Gleevec).51 A complete hemato-
`logical response after decitabine treatment was seen in up to 50% of
`the Gleevec-resistant patients in the chronic phase of the disease.
`Furthermore, the demethylating effect was assessed by methylation
`analysis of LINE-1 elements as well as p15 tumor suppressor gene.
`Surprisingly, the degree of LINE-1 hypomethylation at the end of
`therapy was higher in patients who did not subsequently respond to
`therapy. This has been interpreted to reflect the possibility that
`hypomethylated cells die more rapidly, while cells resistant to therapy
`can withstand higher degrees of hypomethylation.51
`Lastly, it will also be critical to design treatment schedules that
`maximize the reversion of epigenetic mutations with minimal influ-
`ences on epigenetic effects required for normal cellular functions.57
`This is an important aspect in light of the finding that strong and
`continuous demethylation in animal and cellular models have been
`shown to promote genome instability and thereby induce tumor
`formation.58,59 A recent analysis used Apcmin mice with three different
`levels of the DNA maintenance methyltransferase DNMT1 to
`model the degree of hypomethylation observed in tumors (Yamada
`intestinal tumorigenesis but promotes microadenoma formation in
`
`10
`
`Epigenetics
`
`2006; Vol. 1 Issue 1
`
`

`

`azanucleoside derivatives
`
`5-azacytidine (Vidaza)
`5-aza-2´-deoxycytidine
`(Decitabine, Dacogen)
`
`Covalent enzyme trapping (26)
`
`zebularine
`
`Covalent enzyme trapping (33)
`
`small-molecule compounds
`
`hydralazine
`
`noncovalent enzyme inhibition (68)
`
`procaine (procainamide)
`
`binding to CpG-rich DNA (65),
`competitive inhibition
`of DNMT1 (66)
`
`~ ~N
`d o
`
`(
`
`O~N~
`
`..,,.
`
`DNA Methyltransferase Inhibitors
`
`Table 2 Overview of DNA methyltransferase inhibitors
`
`Compound
`
`Proposed Inhibitory Mechanism
`
`Structure
`
`determined, it suggests that epige-
`netic therapies could benefit from a
`further refinement of treatment
`schedules and the use of more specific
`drugs.
`
`DEVELOPMENT OF NOVEL DNA
`METHYLTRANSFERASE
`INHIBITORS
`The apparent lack of specificity
`and the inherent toxicity of nucleo-
`side inhibitors have invigorated the
`search for alternative DNA methyl-
`transferase inhibitors.25 The aim has
`been to identify compounds that do
`not need to be incorporated into
`DNA and thus would circumvent
`the toxic side-effects caused by the
`formation of protein-DNA adducts.61
`Alternatively, it might also be feasible
`to reduce DNA methyltransferase
`activity though oligonucleotides,
`either by allosteric enzyme inhibi-
`tion62 or by siRNA-mediated mRNA
`degradation.63,64
`There are several small-molecule-
`like compounds that have been
`described to induce demethylation
`in human cancer cell lines (Table. 2).
`Interestingly, some of these represent
`established drugs for the treatment
`of diseases not related to cancer. For
`example, the local anaesthetic pro-
`caine and its derivative procainamide,
`an approved antiarrhythmic drug,
`have been described to cause signifi-
`cant demethylation of genomic DNA
`in human cancer cell lines.65,66 This
`was confirmed by drug-induced
`demethylation and reactivation of
`the hypermethylated RARβ2 tumor
`suppressor gene.65 To explain the
`demethylating effects, it was suggest-
`ed that procaine and procainamide
`associate with CpG-rich sequences
`in the genome and thereby interfere
`with the DNA methylation machin-
`ery.65 Additionally, it has also been
`suggested
`that
`procainamide
`decreases the affinity of DNMT1
`for both DNA and the methyl-
`group donor S-adenosyl-L-methio-
`nine.66 Inhibition of DNA methylation has also been reported for
`hydralazine, an established antihypertensive drug that has been
`described to have demethylating activity in human cancer cell
`lines.67 Experimental results derived from gene-specific and
`whole-genome methylation analyses
`suggested
`significant
`drug-induced demethylation that was accompanied by corresponding
`gene reactivation.67,68 Results from a recent phase I of hydralazine in
`
`OH
`
`0
`
`~ '"
`Y'OH
`
`OH
`
`(-)-epigallocatechin-3-gallate
`(EGCG)
`
`noncovalent enzyme
`inhibition (70)
`
`HO
`
`RG108
`
`noncovalent enzyme
`inhibition (73)
`
`the colon. In this context, demethylation of centromeric satellite
`elements might play an important role in chromosomal instability.14
`The analysis of DNA from decitabine-treated MDS patients after
`various treatment time points demonstrated that drug-induced
`hypomethylation also led to demethylation of chromosome 1 satellite
`2 sequences,55 which has been linked to chromosomal instability.60
`While the clinical significance of this observation remains to be
`
`www.landesbioscience.com
`
`Epigenetics
`
`11
`
`

`

`DNA Methyltransferase Inhibitors
`
`cervical cancer patients also indicated demethylation and concomitant
`reactivation of hypermethylated tumor suppressor genes in tumor
`samples.68 While these results seemed impressive at first, it needs