Key words: DNA methylation; DNA hypomethylation; epigenetics; leukemia; hematologic malignancy; CpG island; demethylation; chemotherapeutics; AML; MDS; CLL; p16

Abstract

The DNA methylation profile of cancer cells is frequently characterized by global hypomethylation and simultaneous hypermethylation of selected CpG island gene promoters. In recent years the epigenetic phenomenon of DNA promoter methylation has gained increasing recognition as an important mechanism for transcriptional inactivation of cancer related genes. Studies on both liquid and solid tumors have revealed myriad aberrant methylation events, some of which may provide important clues to the pathogenesis of these tumors. The identification of these methylation alterations and elucidation of the mechanistic events surrounding them are of prime importance, as the methylation status of cancer cells can now be manipulated in vivo with demethylating chemotherapeutics.

INTRODUCTION

Without question genetic alterations underlie the pathogenesis of cancer. In recent years, however, epigenetic alterations have gained increasing recognition as important participants in tumor development and progression. Epigenetic changes include DNA methylation and histone modifications (acetylation and methylation), which influence chromatin structure or modify the DNA without altering the native nucleotide sequence. This may be particularly relevant in the leukemias, as mouse models created by insertion of an oncogenic chromosomal translocation fusion protein sometimes fail to produce overt leukemia, thus indicating that additional genetic or epigenetic events are required for malignancy [1;2]. Because epigenetic changes are potentially reversible, they make attractive targets for therapeutic intervention. Therefore, a thorough understanding of epigenetic regulation and the identification of loci involved in dysregulation are critical for the rational use of demethylating agents and histone deacetylase inhibitors in cancer patients. In addition, DNA methylation alterations can also be exploited as biomarkers for monitoring treatment efficacy and minimal residual disease. This review will focus on the current understanding of DNA methylation abnormalities in hematologic malignancies and discuss how this knowledge contributes to our understanding of the pathogenesis of these diseases.

Methylation and transcriptional regulation

2.1 CpG islands

CG dinucleotides are unevenly distributed in the genome. The vast majority are found in repetitive elements and heterochromatin, and in this context they are normally methylated [3]. CpG islands, however, are stretches of DNA 200-2000 basepair long, with a higher frequency of CG dinucleotides than the bulk of the genome [4;5]. When CpG islands are present in the promoter or 5' region of a gene they are usually unmethylated, regardless of the transcriptional state of the gene. Methylation patterns are established during normal embryonic development [6], and participate in X chromosome inactivation [6] and genomic imprinting [7]. Tissue-specific methylation also occurs [8;9], and methylation has been shown to increase in some tissues with aging [10].

Cytosine methylation usually only occurs in the context of a CG dinucleotide. The transfer of a methyl group to the cytosine base is catalyzed by various DNA methyltransferase (DNMT) enzymes. DNMT1, known as the maintenance methyltransferase, acts at the replication fork during DNA synthesis to convert hemi-methylated DNA to a fully methylated state [11]. On the other hand, DNMT3a and DNMT3b possess de novo methyltransferase activity and methylate previously unmethylated loci [12]. Recent evidence, however, suggests that DNMT1 and DNMT3b work together to establish and maintain a hypermethylated phenotype in tumor cells [13].

2.2 Methylation and transcriptional regulation

Methylation of promoter CpG islands is correlated with transcriptional inactivation of the associated gene, and this inactivation is equivalent to a loss-of-function genetic alteration such as a deletion or mutation. If the gene has tumor suppressor properties or is involved in growth regulation, differentiation, or apoptosis, aberrant methylation can play a central role in tumorigenesis (reviewed in [14;15]). Methylation can be biallelic or monoallelic. For example, monoallelic methylation with a concomitant loss-of-function genetic alteration on the other allele results in complete lack of a normal gene product [16]. Alternatively, biallelic methylation can also cause complete transcriptional inactivation [17].
Concurrent with CpG island hypermethylation in tumor cells there is an overall decrease in 5-methylcytosine levels arising from hypomethylation of normally methylated repetitive elements [18;19]. The biologic consequences of this global hypomethylation remain unclear. However, hypomethylation of a normally methylated gene promoter can lead to activation of that gene, as will be discussed later. Some of the biologic processes that are subject to altered methylation in cancer are shown in Figure 1.
Exactly how methylation abrogates transcription remains unclear, but involves recruitment of methyl-binding proteins and co-repressor complexes [20]. These, in turn, may inhibit transcription factor binding and permit adoption of a closed chromatin configuration mediated by histone deacetylation. Transcription from methylated promoters can be induced by treatment with 5-azacytidine or 5-aza-2'-deoxycytidine [21]. These compounds covalently bind DNMT1, resulting in depletion of enzymatic activity. Histone deacetylase inhibitors allow an “open” chromatin configuration and have been shown to be synergistic when used with demethylating drugs in some systems [22;23]. Unraveling the complexities of the interactions between DNA methylation and chromatin modification is critical for understanding their roles in tumor pathogenesis and for designing rational approaches for the use of epigenetic modifiers in cancer patients.

3. Laboratory methods for detecting methylation

Standard nucleotide sequencing does not discriminate between cytosine and 5-methylcytosine, so alternative methods must be employed to assess the presence or absence of methylation. Methylation-sensitive restriction enzymes are frequently used, often in conjunction with their methylation-insensitive isoschizomers (e.g. HpaII and MspI), followed by hybridization with a gene-specific probe. Differential digestion when using these two enzymes provides a quantitative measure of the amount of methylation in the restriction site being examined. A more recent innovation is the use of sodium bisulfite to convert unmethylated cytosines to uracil (and then to thymine in subsequent PCR reactions), while leaving methylated cytosines unchanged [24]. This conversion is the cornerstone of bisulfite genomic sequencing, which allows one to examine each CG dinucleotide within a PCR amplicon, and methylation-specific polymerase chain reaction (MS-PCR), which provides a readout of the presence or absence of methylated alleles depending on the specificity of the primers used [25].
While these techniques provide important information on selected candidate genes, they do not allow the assessment of global levels of promoter methylation, nor can they be used to identify novel methylated sequences. Our laboratory uses restriction landmark genomic scanning (RLGS) to assay up to 2000 methylation-sensitive restriction sites in a single tumor profile [26]. RLGS is not dependent on hybridization kinetics, and prior knowledge of specific sequences is not needed in order to detect methylation changes (reviewed in [27]). A detailed discussion of techniques utilized in methylation analysis is outside the scope of this review, and the reader is referred to a number of excellent articles on this subject [28-30].

4. Methylation in normal hematopoiesis

As mentioned previously, not all promoter methylation is abnormal or pathogenic. In fact, dynamic changes in promoter methylation and chromatin structure appear to be important for expression of growth factors, growth factor receptors, cytokines, and other molecules during normal myeloid development [31]. In B cells, demethylation of one kappa light-chain allele precedes somatic rearrangement of that allele, and retention of methylation on the germ line allele may be one mechanism for preventing two somatic rearrangements in the same cell [32]. Differential methylation patterns have been correlated with regulation of expression of PU.1 in several blood cell lineages [33]. Intron 2 of the M-CSF gene is unmethylated and the gene is expressed in monocytes and macrophages, while non-expressing granulocytes and lymphocytes are methylated at this locus [34]. Similarly, the G-CSF receptor is unmethylated in normal granulocytes and monocytes but methylated in lymphocytes [35]. Methylation also plays a key role in regulating gene expression in T cells (reviewed in [36]). Demethylation of IFN-gamma is associated with increased expression in activated mouse CD8+ T cells [37]. Pestano and colleagues examined CD8+ T cells that fail to recognize the MHC class I molecule and inadvertently escape the thymus. Once in the periphery, these cells have progressive methylation of the CD8 gene, followed by decreased CD8 expression and upregulation of Fas and FasL, leading them to succumb to apoptosis [38]. In another study, clones derived from single T cells of an individual showed marked variability in the methylation patterns, indicating that there might be more heterogeneity in methylation patterns than previously appreciated [39]

It is important to understand methylation patterns in normal tissues in order to accurately interpret abnormalities in cancer. This can be problematic when using highly sensitive techniques such as MS-PCR to evaluate the presence of methylated alleles in a tumor sample. MS-PCR can detect methylation in one cell out of 1000 [40]. Because tumors samples inevitably contain some normal tissue contamination, the presence of low-level methylation could reflect normally methylated alleles in noncancerous blood cells or stroma. Cancer-related genes that show some degree of methylation in normal hematopoietic cells include p15 [41-43], p21 [44], and IGF-2 [45;46].

5. Hypermethylation in myeloid leukemia and myelodysplastic syndrome

5.1 Acute myeloid leukemia

Several groups have shown that acute myeloid leukemia (AML) cells possess a number of methylation lesions. Melki et al. analyzed promoter methylation of eight genes by bisulfite genomic sequencing [47]. They found that 19 of 20 (95%) of AML patients were hypermethylated for at least one gene, and 15 patients (75%) had hypermethylation of at least two genes. There was no correlation between the degree of methylation and methyltransferase levels in these patients, although previous work by this group had shown increased DMNT1 expression in some AML patients [48]. In a somewhat larger study, Toyota and colleagues examined 15 promoters in 36 AML patients and found high levels of methylation for a subset of these genes [49]. Interestingly, there was an inverse correlation between the number of methylated promoters and the age of the patient, i.e. older patients had statistically significant fewer methylated genes. Our group used RLGS to examine 16 paired AML diagnostic and remission samples and discovered a wide variation in the amount of aberrant methylation among different patients [50]. These changes occur in a non-random fashion, and sequence analysis of methylated loci allowed us to identify many novel targets of methylation. We also demonstrated an increased number of methylated loci located on chromosome 11 [51]. De Bustros et al. had previously identified 11p as a methylation “hot spot” [52] in multiple types of neoplasms, and our study allowed us to extend this phenomenon to the entire chromosome. Taken together, these studies suggest that AML frequently demonstrates a hypermethylated phenotype, and underscore the marked heterogeneity that exists between patients.
Some of the most frequently examined genes in AML have been the p15 and p16 cyclin-dependent kinase inhibitors. Cameron et al. found variable levels of p15 methylation [53]. In this study transcriptional repression was correlated more strongly with the overall density of methylated sites rather than with any specific sites in the promoter. Other groups have also shown that p15 methylation is quite variable between individual patients and even among different cells within the same patient [54;55]. But while p15 is often methylated in AML, p16 methylation is much less frequently detected [56-58]. Indeed, Herman et al. reported that AML is characterized by p15 methylation in the absence of p16 methylation, while high-grade non-Hodgkin’s lymphomas often display methylation of p16 but not p15, thus setting the stage for classification of hematologic malignancies according to their methylation profiles [59]. Other genes with a high frequency of methylation in AML include the estrogen receptor (ER) [60-62], E-cadherin [63-65], and HIC1 [66-68].
But what do these methylation patterns tell us about the biology of AML? HIC1 methylation appears to occur in late-stage AML [69], and WIT1 methylation is associated with chemoresistant AML [70]. Elegant work by Di Croce et al. has provided new insight into how oncogenic fusion proteins, a hallmark of AML, might interact with the methylation machinery to disrupt transcription [71]. They showed that the PML-RAR fusion protein could recruit methyltransferases to the RARâ2 promoter, inducing methylation-mediated transcriptional repression. Although they examined only one target promoter, this work has important implications for leukemogenesis. Do other fusion proteins act in a similar fashion achieve transcriptional repression? If so, identification of these targets will provide clues to the pathogenesis of these diseases. Importantly, methylation of specific, rather than random, targets may underlie the observation that different hematologic malignancies harbor distinct methylation signatures [72].

5.2 Chronic myeloid leukemia

Chronic myeloid leukemia (CML) can be divided into three stages: chronic phase, accelerated phase, and a terminal blast crisis [73]. Therefore, several investigators have examined stage-specific methylation events in order to elucidate the molecular mechanisms responsible for disease progression. Increasing levels of methylation of the calcitonin [74;75], HIC1 [76], ER [77], and ABL1 [78] genes have all been found during evolution from chronic phase to blast crisis. Other stage-specific events include loss-of-imprinting of IGF2 (see later). Aberrant methylation has also been shown to occur around the major breakpoint cluster region in CML patients both with and without the Philadelphia chromosome [79].

5.3 Myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is included here because of the tendency for MDS to progress to AML. As was reported for AML, MDS showed a lack of p16 methylation, but p15 methylation was present in 16 of 32 patients (50%) [80]. Furthermore, the frequency of p15 methylation was greater in high-risk MDS and increased in some patients as they progressed to overt leukemia. Similar results were reported in another study in which p15 methylation was followed serially as patients progressed through their disease [81].

6. Hypermethylation in lymphoproliferative disorders

6.1 Acute lymphocytic leukemia

Many of the same genes that are methylated in AML are also methylated in acute lymphocytic leukemia (ALL). For example, investigators have shown variable levels of p15 [82], ER [83], HIC1 [84] and E-cadherin [85] methylation in primary ALL samples of both B and T cell lineages. In addition, hypermethylation of p73, a homologue of p53, is methylated in a number of ALL cell lines [86;87].
Recently, Roman-Gomez et al. examined p21 methylation in 124 adult and pediatric ALL cases, including both B-ALL and T-ALL [88]. p21 is a cyclin-dependent kinase inhibitor and candidate tumor suppressor gene that is frequently down-regulated in ALL. They found that promoter methylation was highly correlated with transcriptional repression. Furthermore, patients with p21 methylation had significantly reduced disease-free survival and overall survival times, and the methylation status was an independent prognostic factor for prediction of disease-free survival.

6.2 Chronic lymphocytic leukemia

Few studies have examined hypermethylation events in chronic lymphocytic leukemia (CLL). Bechter et al. reported that methylation of hTERT, the catalytic subunit of telomerase, is correlated with decreased levels of telomerase activity in CLL [89]. Biallelic methylation of the DXS255 polymorphic locus on the X chromosome was found in one study of female CLL patients [90], and methylation of E-cadherin was described in three of five patients with CLL [91]. Hypomethylation events have also been examined and these may play an important role in the pathogenesis of CLL (see later).

Lymphoma

Esteller and colleagues have recently correlated MGMT promoter methylation and improved survival in diffuse large B-cell lymphoma [92]. MGMT repairs DNA damage caused by alkylating agents such as cyclophosphamide, which is frequently used in chemotherapeutic regimens for the treatment of lymphoma. Therefore, the authors hypothesize that methylation-induced silencing of MGMT may inhibit the ability of the neoplastic cells to repair damage caused by aklyating agents.
Sui et al. have examined the methylation status of a panel of genes in 33 cases of natural killer cell lymphoma and found high levels of p73 methylation (94%) as well as MLH1 (63%), p16 (63%), p15 (48%), and RARβ (47%) [93]. Of note, they described methylation of two or more genes in 88% of these cases, and described differential methylation between the primary and metastatic lesions in 2 cases.
Methylation of DAP-kinase, a regulator of apoptosis, has been reported in Burkitt’s lymphoma and B-cell lymphoma [94]. In this study, down-regulation of DAP-kinase transcription was associated with increased resistance to IFNγ mediated apoptosis. Other genes hypermethylated in various types of lymphoma include p15 and p16 [95], ER [96], HIC1 [97], and Myf-3 [98].

6.4 Multiple myeloma

Cyclin-dependent kinase genes p16 and p15 have been shown to be targets of methylation in both multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) [99-101]. Mateos et al. found p16 methylation in 41 of 98 cases of MM and 4 of 5 cases of plasma cell leukemia, but not in MGUS, concluding that p16 methylation was associated with advanced or aggressive disease [99]. On the other hand, Guillerm and colleagues examined p15 and p16 methylation in 33 cases each of MM and MGUS and found few differences between the two groups [102]. DAP-kinase methylation has also been reported in MM and MGUS [103]. Clearly, more work is needed to determine the role that methylation plays in the pathogenesis and progression of MM and MGUS.

7. Imprinting and leukemia

Genomic imprinting is a normal process that results in parent-of-origin allele-specific transcription, mediated to a large extent by DNA methylation. In most tissues IGF2 is expressed only from the paternal allele. However, several groups have observed demethylation and biallelic expression from neoplastic cells in MDS [104], CML [105], and AML [106]. Originally it was thought that this change represented a specific alteration in tumor cells. However, it has now been shown by several groups that loss of imprinting of IGF2 and subsequent biallelic expression is a feature of some normal hematopoietic cells when they are in a proliferative state [107;108]. Methylation of the CTCF-binding site in the H19/IGF2 locus can also be responsible for disruption of normal imprinting in cancer [109;110]. Recently it was reported that the normally imprinted neuronatin gene, NNAT, undergoes biallelic methylation in childhood leukemias [111]. Since imprinted genes are frequently involved in growth regulatory functions, a more complete understanding of both normal and tumor-associated methylation changes is needed. The readers are referred to a recent review of imprinting and cancer for further details [112].

8. Hypomethylation in hematologic malignancies

Just as hypermethylation events can lead to transcriptional repression of tumor suppressor genes, hypomethylation, or demethylation, can activate potential oncogenes. The TCL1 gene is oncogenic in T-cell prolymphocytic leukemia following translocation to the T-cell receptor and subsequent over-expression. However, some cases of Burkitt’s lymphoma (BL) and CLL exhibit TCL1 over-expression without rearrangement of TCL1. Yuille et al. reported biallelic promoter demethylation in BL cell lines and primary CLL samples which correlated with over-expression of TCL1, suggesting that hypomethylation events in these neoplasms could be functionally equivalent to oncogenic translocations [113].
Similarly, the HOX11 gene is translocated and over-expressed in some cases of T-ALL, but over-expression also occurs in the absence of translocations. Demethylation of the HOX11 promoter was then discovered to be an alternative to rearrangement for activation of this oncogene [114]. Several groups have investigated hypomethylation events in CLL, including demethylation of the multidrug resistance gene, MRD1 [115], ornithine decarboxylase [116], Erb-A1 [117], and the anti-apoptotic gene BCL2 [118].
In contrast to localized, site-specific CpG island hypermethylation events that occur in tumors, many cancers have an overall decrease of 5-methylcytosine [119;120]. Wahlfors et al. confirmed this observation in CLL and reported that this disease is characterized by global hypomethylation [121]. The significance of tumor-associated global hypomethylation and its relationship to CpG island hypermethylation remains unclear, however it is another indication of a generalized defect in the regulation of the methylation machinery.

9. Therapeutic considerations

Methylation and chromatin modifying agents are currently being evaluated in clinical trials. Therefore, it is critical that we more fully understand the spectrum of epigenetic features in both normal and tumor cells. 5-aza-2'-deoxycitidine has been used to treat various tumors and appears to exert both demethylating and cytotoxic effects (reviewed in [122]). Patients with MDS have shown overall response rates of 49% [123], and in one trial 5-aza-2'-deoxycitidine was associated with major cytogenetic responses and improved survival [124]. Depsipeptide (FR901228), a histone deacetylase inhibitor, has demonstrated efficacy against cutaneous and peripheral T-cell lymphomas [125]. Combined use of demethylating agents and histone deacetylase inhibitors may result in even more promising results. Suzuki et al. recently reported a number of genes whose regulation in colorectal cancer cell lines can be modulated by 5-aza-2'-deoxycytidine, trichostatin A (a histone deacetylase inhibitor) or a combination of both drugs [126]. This work increases our understanding of the widespread effects of these drugs and provides the groundwork for mechanistic investigation into the regulation these genes.
In addition to demethylating agents and histone deacetylase inhibitors, antisense oligonucleotides directed against DNA methyltransferases are also in clinical trials. One such compound, MG-98, is currently being used in Phase I trials for patients with AML and Phase II trials for patients with epithelial tumors [127].
Importantly, we need to develop appropriate markers and standardized methods for evaluating treatment efficacy and monitoring minimal residual disease in patients who receive epigenetic modifying drugs. Given the plethora of hypermethylation targets in hematologic malignancies, how does one choose which promoters to evaluate to determine the efficacy of a demethylating agent? How will we quantify the level of demethylation of a target gene so that data from different investigators can be reasonably compared? Will demethylation of tumor suppressor CpG islands and re-establishment of transcription result in inadvertent activation of oncogenes and loss of imprinting? These questions and others will need to be addressed in order to make the best use of these therapeutic agents and optimize their use in targeting epigenetic lesions.
Figure 2 illustrates some of the possible consequences of using a demethylating agent in a leukemic patient. This therapy can result in cytotoxicity, differentiation, and/or demethylation of the leukemic cells. Investigators must assess the level of demethylation, determine if global or gene-specific hypomethylation has detrimental effects on the patient, and monitor for re-methylation of demethylated genes. One must consider which genes to evaluate in order to document that sufficient demethylation has occurred. Ideally, these genes would be completely unmethylated in normal tissues in order to avoid a positive signal obtained by very sensitive PCR methods. As noted earlier, some genes exhibit considerable amounts of methylation in normal tissues, thus rendering them unsuitable for monitoring treatment efficacy unless assayed in a quantitative fashion.

10. Conclusions

Most hematologic malignancies appear to have some degree of epigenetic dysregulation. While lesions such as p15 methylation are found in the majority of the diseases examined, others, such as p16 methylation, seem to be more specific for a certain type of disease [128]. Our group has described a number of leukemia-specific methylation targets that are not found in solid tumors [129]. However, with the increasing number of methylated genes being reported, the biologic and clinical significance of many of these events remains undetermined. It is unlikely that each of these methylation alterations has a functional role in the pathogenesis of the particular tumor in which it was found. The challenge for molecular biologists and clinicians is to tease out the relevant targets that can be exploited for diagnostic and therapeutic purposes. The analysis of larger sample sets and the further development of high-throughput methylation scans [130] should greatly aid in deciphering the molecular pathogenesis of leukemias and lymphomas, as well as provide useful tools for subclassification and prognostication.

Acknowledgements
The authors apologize to all those whose works could not be cited due to space limitations. We are indebted to Dominic J. Smiraglia and Guido Marcucci for helpful discussions and critical review of the manuscript. This work was supported by NIH grants CA089317 (L.R.) and CA93548 (C.P.). L.R. was also partially supported by NIH postdoctoral training grant T32-CA09338.

References
[1] K.L.Rhoades, C.J.Hetherington, N.Harakawa, D.A.Yergeau, L.Zhou, L.Q.Liu, M.T.Little, D.G.Tenen, D.E.Zhang, Analysis of the role of AML1-ETO in leukemogenesis, using an inducible transgenic mouse model, Blood 96 (2000) 2108-2115.
[2] L.H.Castilla, L.Garrett, N.Adya, D.Orlic, A.Dutra, S.Anderson, J.Owens, M.Eckhaus, D.Bodine, P.P.Liu, The fusion gene Cbfb-MYH11 blocks myeloid differentiation and predisposes mice to acute myelomonocytic leukaemia [letter], Nat.Genet. 23 (1999) 144-146.
[3] A.P.Bird, CpG-rich islands and the function of DNA methylation, Nature 321 (1986) 209-213.
[4] M.Gardiner-Garden, M.Frommer, CpG islands in vertebrate genomes, J.Mol.Biol. 196 (1987) 261-282.
[5] D.Takai, P.A.Jones, Comprehensive analysis of CpG islands in human chromosomes 21 and 22, Proc.Natl.Acad.Sci.U.S.A 99 (2002) 3740-3745.
[6] A.Razin, T.Kafri, DNA methylation from embryo to adult, Prog.Nucleic Acid Res.Mol.Biol. 48 (1994) 53-81.
[7] A.Razin, H.Cedar, DNA methylation and genomic imprinting, Cell 77 (1994) 473-476.
[8] D.J.Grant, H.Shi, C.T.Teng, Tissue and site-specific methylation correlates with expression of the mouse lactoferrin gene, J.Mol.Endocrinol. 23 (1999) 45-55.
[9] M.A.Gama-Sosa, R.M.Midgett, V.A.Slagel, S.Githens, K.C.Kuo, C.W.Gehrke, M.Ehrlich, Tissue-specific differences in DNA methylation in various mammals, Biochim.Biophys.Acta 740 (1983) 212-219.
[10] N.Ahuja, Q.Li, A.L.Mohan, S.B.Baylin, J.P.Issa, Aging and DNA methylation in colorectal mucosa and cancer, Cancer Res. 58 (1998) 5489-5494.
[11] M.R.Rountree, K.E.Bachman, S.B.Baylin, DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci, Nat.Genet. 25 (2000) 269-277.
[12] M.Okano, S.Xie, E.Li, Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases, Nat.Genet. 19 (1998) 219-220.
[13] I.Rhee, K.E.Bachman, B.H.Park, K.W.Jair, R.W.Yen, K.E.Schuebel, H.Cui, A.P.Feinberg, C.Lengauer, K.W.Kinzler, S.B.Baylin, B.Vogelstein, DNMT1 and DNMT3b cooperate to silence genes in human cancer cells, Nature 416 (2002) 552-556.
[14] J.F.Costello, C.Plass, Methylation matters, J.Med.Genet. 38 (2001) 285-303.
[15] S.B.Baylin, J.G.Herman, DNA hypermethylation in tumorigenesis: epigenetics joins genetics, Trends Genet. 16 (2000) 168-174.
[16] M.Esteller, M.F.Fraga, M.Guo, J.Garcia-Foncillas, I.Hedenfalk, A.K.Godwin, J.Trojan, C.Vaurs-Barriere, Y.J.Bignon, S.Ramus, J.Benitez, T.Caldes, Y.Akiyama, Y.Yuasa, V.Launonen, M.J.Canal, R.Rodriguez, G.Capella, M.A.Peinado, A.Borg, L.A.Aaltonen, B.A.Ponder, S.B.Baylin, J.G.Herman, DNA methylation patterns in hereditary human cancers mimic sporadic tumorigenesis, Hum.Mol.Genet. 10 (2001) 3001-3007.
[17] M.L.Veigl, L.Kasturi, J.Olechnowicz, A.H.Ma, J.D.Lutterbaugh, S.Periyasamy, G.M.Li, J.Drummond, P.L.Modrich, W.D.Sedwick, S.D.Markowitz, Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers, Proc.Natl.Acad.Sci.U.S.A 95 (1998) 8698-8702.
[18] M.A.Gama-Sosa, V.A.Slagel, R.W.Trewyn, R.Oxenhandler, K.C.Kuo, C.W.Gehrke, M.Ehrlich, The 5-methylcytosine content of DNA from human tumors, Nucleic Acids Res. 11 (1983) 6883-6894.
[19] A.P.Feinberg, C.W.Gehrke, K.C.Kuo, M.Ehrlich, Reduced genomic 5-methylcytosine content in human colonic neoplasia, Cancer Res. 48 (1988) 1159-1161.
[20] P.L.Jones, G.J.Veenstra, P.A.Wade, D.Vermaak, S.U.Kass, N.Landsberger, J.Strouboulis, A.P.Wolffe, Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription, Nat.Genet. 19 (1998) 187-191.
[21] P.A.Jones, S.M.Taylor, T.Mohandas, L.J.Shapiro, Cell cycle-specific reactivation of an inactive X-chromosome locus by 5- azadeoxycytidine, Proc.Natl.Acad.Sci.U.S.A 79 (1982) 1215-1219.
[22] E.E.Cameron, K.E.Bachman, S.Myohanen, J.G.Herman, S.B.Baylin, Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer, Nat.Genet. 21 (1999) 103-107.
[23] H.Suzuki, E.Gabrielson, W.Chen, R.Anbazhagan, M.Van Engeland, M.P.Weijenberg, J.G.Herman, S.B.Baylin, A genomic screen for genes upregulated by demethylation and histone deacetylase inhibition in human colorectal cancer, Nat.Genet. (2002).
[24] S.J.Clark, J.Harrison, C.L.Paul, M.Frommer, High sensitivity mapping of methylated cytosines, Nucleic Acids Research 22 (1994) 2990-2997.
[25] J.G.Herman, J.G.Graff, S.Myohanen, B.D.Nelkin, S.B.Baylin, Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands, Proc.Natl.Acad.Sci.U.S.A 93 (1996) 9821-9826.
[26] I.Hatada, Y.Hayashizaki, S.Hirotsune, H.Komatsubara, T.Mukai, A genomic scanning method for higher organisms using restriction sites as landmarks, Proc.Natl.Acad.Sci.U.S.A 88 (1991) 9523-9527.
[27] L.J.Rush, C.Plass. Restriction landmark genomic scanning for DNA methylation in cancer: Past, present, and future applications. Anal.Biochem. 2002. Ref Type: In Press
[28] E.J.Oakeley, DNA methylation analysis: a review of current methodologies, Pharmacol.Ther. 84 (1999) 389-400.
[29] H.H.Dahl, W.M.Hutchison, Analysis of in vivo methylation, Methods Mol.Biol. 130 (2000) 47-57.
[30] M.C.Fruhwald, C.Plass, Global and gene-specific methylation patterns in cancer: aspects of tumor biology and clinical potential, Mol.Genet.Metab 75 (2002) 1-16.
[31] M.Lubbert, R.Mertelsmann, F.Herrmann, Cytosine methylation changes during normal hematopoiesis and in acute myeloid leukemia, Leukemia 11 Suppl 1 (1997) S12-S18.
[32] R.Mostoslavsky, N.Singh, A.Kirillov, R.Pelanda, H.Cedar, A.Chess, Y.Bergman, Kappa chain monoallelic demethylation and the establishment of allelic exclusion, Genes Dev. 12 (1998) 1801-1811.
[33] L.Amaravadi, M.J.Klemsz, DNA methylation and chromatin structure regulate PU.1 expression, DNA Cell Biol. 18 (1999) 875-884.
[34] J.Felgner, H.Kreipe, K.Heidorn, K.Jaquet, R.Heuss, F.Zschunke, H.J.Radzun, M.R.Parwaresch, Lineage-specific methylation of the c-fms gene in blood cells and macrophages, Leukemia 6 (1992) 420-425.
[35] J.Felgner, K.Heidorn, D.Korbacher, S.O.Frahm, R.Parwaresch, Cell lineage specificity in G-CSF receptor gene methylation, Leukemia 13 (1999) 530-534.
[36] O.Avni, A.Rao, T cell differentiation: a mechanistic view, Curr.Opin.Immunol. 12 (2000) 654-659.
[37] D.R.Fitzpatrick, K.M.Shirley, L.E.McDonald, H.Bielefeldt-Ohmann, G.F.Kay, A.Kelso, Distinct methylation of the interferon gamma (IFN-gamma) and interleukin 3 (IL-3) genes in newly activated primary CD8+ T lymphocytes: regional IFN-gamma promoter demethylation and mRNA expression are heritable in CD44(high)CD8+ T cells, J.Exp.Med. 188 (1998) 103-117.
[38] G.A.Pestano, Y.Zhou, L.A.Trimble, J.Daley, G.F.Weber, H.Cantor, Inactivation of misselected CD8 T cells by CD8 gene methylation and cell death, Science 284 (1999) 1187-1191.
[39] X.Zhu, C.Deng, R.Kuick, R.Yung, B.Lamb, J.V.Neel, B.Richardson, S.Hanash, Analysis of human peripheral blood T cells and single-cell-derived T cell clones uncovers extensive clonal CpG island methylation heterogeneity throughout the genome, Proc.Natl.Acad.Sci.U.S.A 96 (1999) 8058-8063.
[40] J.G.Herman, J.G.Graff, S.Myohanen, B.D.Nelkin, S.B.Baylin, Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands, Proc.Natl.Acad.Sci.U.S.A 93 (1996) 9821-9826.
[41] K.Sakashita, K.Koike, T.Kinoshita, M.Shiohara, T.Kamijo, S.Taniguchi, T.Kubota, Dynamic DNA methylation change in the CpG island region of p15 during human myeloid development, J.Clin.Invest 108 (2001) 1195-1204.
[42] E.E.Cameron, S.B.Baylin, J.G.Herman, p15(INK4B) CpG island methylation in primary acute leukemia is heterogeneous and suggests density as a critical factor for transcriptional silencing, Blood 94 (1999) 2445-2451.
[43] A.Aggerholm, P.Guldberg, M.Hokland, P.Hokland, Extensive intra- and interindividual heterogeneity of p15INK4B methylation in acute myeloid leukemia, Cancer Res. 59 (1999) 436-441.
[44] J.Roman-Gomez, J.A.Castillejo, A.Jimenez, M.G.Gonzalez, F.Moreno, M.C.Rodriguez, M.Barrios, J.Maldonado, A.Torres, 5' CpG island hypermethylation is associated with transcriptional silencing of the p21(CIP1/WAF1/SDI1) gene and confers poor prognosis in acute lymphoblastic leukemia, Blood 99 (2002) 2291-2296.
[45] I.M.Morison, M.R.Eccles, A.E.Reeve, Imprinting of insulin-like growth factor 2 is modulated during hematopoiesis, Blood 96 (2000) 3023-3028.
[46] W.K.Hofmann, S.Takeuchi, M.A.Frantzen, D.Hoelzer, H.P.Koeffler, Loss of genomic imprinting of insulin-like growth factor 2 is strongly associated with cellular proliferation in normal hematopoietic cells, Exp.Hematol. 30 (2002) 318-323.
[47] J.R.Melki, P.C.Vincent, S.J.Clark, Concurrent DNA hypermethylation of multiple genes in acute myeloid leukemia, Cancer Res. 59 (1999) 3730-3740.
[48] J.R.Melki, P.Warnecke, P.C.Vincent, S.J.Clark, Increased DNA methyltransferase expression in leukaemia, Leukemia 12 (1998) 311-316.
[49] M.Toyota, K.J.Kopecky, M.O.Toyota, K.W.Jair, C.L.Willman, J.P.Issa, Methylation profiling in acute myeloid leukemia, Blood 97 (2001) 2823-2829.
[50] L.J.Rush, Z.Dai, D.J.Smiraglia, X.Gao, F.A.Wright, M.Fruhwald, J.F.Costello, W.A.Held, L.Yu, R.Krahe, J.E.Kolitz, C.D.Bloomfield, M.A.Caligiuri, C.Plass, Novel methylation targets in de novo acute myeloid leukemia with prevalence of chromosome 11 loci, Blood 97 (2001) 3226-3233.
[51] L.J.Rush, Z.Dai, D.J.Smiraglia, X.Gao, F.A.Wright, M.Fruhwald, J.F.Costello, W.A.Held, L.Yu, R.Krahe, J.E.Kolitz, C.D.Bloomfield, M.A.Caligiuri, C.Plass, Novel methylation targets in de novo acute myeloid leukemia with prevalence of chromosome 11 loci, Blood 97 (2001) 3226-3233.
[52] A.de Bustros, B.D.Nelkin, A.Silverman, G.Ehrlich, B.Poiesz, S.B.Baylin, The short arm of chromosome 11 is a "hot spot" for hypermethylation in human neoplasia, Proc.Natl.Acad.Sci.U.S.A 85 (1988) 5693-5697.
[53] E.E.Cameron, S.B.Baylin, J.G.Herman, p15(INK4B) CpG island methylation in primary acute leukemia is heterogeneous and suggests density as a critical factor for transcriptional silencing, Blood 94 (1999) 2445-2451.
[54] J.E.Dodge, A.F.List, B.W.Futscher, Selective variegated methylation of the p15 CpG island in acute myeloid leukemia, Int.J.Cancer 78 (1998) 561-567.
[55] J.E.Dodge, C.Munson, A.F.List, KG-1 and KG-1a model the p15 CpG island methylation observed in acute myeloid leukemia patients, Leuk.Res. 25 (2001) 917-925.
[56] J.E.Dodge, A.F.List, B.W.Futscher, Selective variegated methylation of the p15 CpG island in acute myeloid leukemia, Int.J.Cancer 78 (1998) 561-567.
[57] J.E.Dodge, C.Munson, A.F.List, KG-1 and KG-1a model the p15 CpG island methylation observed in acute myeloid leukemia patients, Leuk.Res. 25 (2001) 917-925.
[58] M.Toyota, K.J.Kopecky, M.O.Toyota, K.W.Jair, C.L.Willman, J.P.Issa, Methylation profiling in acute myeloid leukemia, Blood 97 (2001) 2823-2829.
[59] J.G.Herman, C.I.Civin, J.P.Issa, M.I.Collector, S.J.Sharkis, S.B.Baylin, Distinct patterns of inactivation of p15INK4B and p16INK4A characterize the major types of hematological malignancies, Cancer Res. 57 (1997) 837-841.
[60] J.P.Issa, B.A.Zehnbauer, C.I.Civin, M.I.Collector, S.J.Sharkis, N.E.Davidson, S.H.Kaufmann, S.B.Baylin, The estrogen receptor CpG island is methylated in most hematopoietic neoplasms, Cancer Res. 56 (1996) 973-977.
[61] Q.Li, K.J.Kopecky, A.Mohan, C.L.Willman, F.R.Appelbaum, J.K.Weick, J.P.Issa, Estrogen receptor methylation is associated with improved survival in adult acute myeloid leukemia, Clin.Cancer Res. 5 (1999) 1077-1084.
[62] J.R.Melki, P.C.Vincent, S.J.Clark, Concurrent DNA hypermethylation of multiple genes in acute myeloid leukemia, Cancer Res. 59 (1999) 3730-3740.
[63] J.R.Melki, P.C.Vincent, S.J.Clark, Concurrent DNA hypermethylation of multiple genes in acute myeloid leukemia, Cancer Res. 59 (1999) 3730-3740.
[64] J.R.Melki, P.C.Vincent, R.D.Brown, S.J.Clark, Hypermethylation of E-cadherin in leukemia, Blood 95 (2000) 3208-3213.
[65] P.G.Corn, B.D.Smith, E.S.Ruckdeschel, D.Douglas, S.B.Baylin, J.G.Herman, E-cadherin expression is silenced by 5' CpG island methylation in acute leukemia, Clin.Cancer Res. 6 (2000) 4243-4248.
[66] J.R.Melki, P.C.Vincent, S.J.Clark, Concurrent DNA hypermethylation of multiple genes in acute myeloid leukemia, Cancer Res. 59 (1999) 3730-3740.
[67] J.P.Issa, B.A.Zehnbauer, S.H.Kaufmann, M.A.Biel, S.B.Baylin, HIC1 hypermethylation is a late event in hematopoietic neoplasms, Cancer Res. 57 (1997) 1678-1681.
[68] J.R.Melki, P.C.Vincent, S.J.Clark, Cancer-specific region of hypermethylation identified within the HIC1 putative tumour suppressor gene in acute myeloid leukaemia, Leukemia 13 (1999) 877-883.
[69] J.P.Issa, B.A.Zehnbauer, S.H.Kaufmann, M.A.Biel, S.B.Baylin, HIC1 hypermethylation is a late event in hematopoietic neoplasms, Cancer Res. 57 (1997) 1678-1681.
[70] C.Plass, F.Yu, L.Yu, M.P.Strout, W.El Rifai, E.Elonen, S.Knuutila, G.Marcucci, D.C.Young, W.A.Held, C.D.Bloomfield, M.A.Caligiuri, Restriction landmark genome scanning for aberrant methylation in primary refractory and relapsed acute myeloid leukemia; involvement of the WIT-1 gene, Oncogene 18 (1999) 3159-3165.
[71] L.Di Croce, V.A.Raker, M.Corsaro, F.Fazi, M.Fanelli, M.Faretta, F.Fuks, C.F.Lo, T.Kouzarides, C.Nervi,
S.Minucci, P.G.Pelicci, Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor, Science 295 (2002) 1079-1082.
[72] J.G.Herman, C.I.Civin, J.P.Issa, M.I.Collector, S.J.Sharkis, S.B.Baylin, Distinct patterns of inactivation of p15INK4B and p16INK4A characterize the major types of hematological malignancies, Cancer Res. 57 (1997) 837-841.
[73] M.J.Mauro, B.J.Druker, Chronic myelogenous leukemia, Curr.Opin.Oncol. 13 (2001) 3-7.
[74] B.D.Nelkin, D.Przepiorka, P.J.Burke, E.D.Thomas, S.B.Baylin, Abnormal methylation of the calcitonin gene marks progression of chronic myelogenous leukemia, Blood 77 (1991) 2431-2434.
[75] K.I.Mills, B.A.Guinn, V.A.Walsh, A.K.Burnett, Increasing methylation of the calcitonin gene during disease progression in sequential samples from CML patients, Leuk.Res. 20 (1996) 771-775.
[76] J.P.Issa, B.A.Zehnbauer, S.H.Kaufmann, M.A.Biel, S.B.Baylin, HIC1 hypermethylation is a late event in hematopoietic neoplasms, Cancer Res. 57 (1997) 1678-1681.
[77] J.P.Issa, B.A.Zehnbauer, C.I.Civin, M.I.Collector, S.J.Sharkis, N.E.Davidson, S.H.Kaufmann, S.B.Baylin, The estrogen receptor CpG island is methylated in most hematopoietic neoplasms, Cancer Res. 56 (1996) 973-977.
[78] F.A.Asimakopoulos, P.J.Shteper, S.Krichevsky, E.Fibach, A.Polliack, E.Rachmilewitz, Y.Ben Neriah, D.Ben Yehuda, ABL1 methylation is a distinct molecular event associated with clonal evolution of chronic myeloid leukemia, Blood 94 (1999) 2452-2460.
[79] C.E.Litz, J.A.Vos, C.M.Copenhaver, Aberrant methylation of the major breakpoint cluster region in chronic myeloid leukemia, Blood 88 (1996) 2241-2249.
[80] T.Uchida, T.Kinoshita, H.Nagai, Y.Nakahara, H.Saito, T.Hotta, T.Murate, Hypermethylation of the p15INK4B gene in myelodysplastic syndromes, Blood 90 (1997) 1403-1409.
[81] H.F.Tien, J.H.Tang, W.Tsay, M.C.Liu, F.Y.Lee, C.H.Wang, Y.C.Chen, M.C.Shen, Methylation of the p15(INK4B) gene in myelodysplastic syndrome: it can be detected early at diagnosis or during disease progression and is highly associated with leukaemic transformation, Br.J.Haematol. 112 (2001) 148-154.
[82] E.E.Cameron, S.B.Baylin, J.G.Herman, p15(INK4B) CpG island methylation in primary acute leukemia is heterogeneous and suggests density as a critical factor for transcriptional silencing, Blood 94 (1999) 2445-2451.
[83] J.P.Issa, B.A.Zehnbauer, C.I.Civin, M.I.Collector, S.J.Sharkis, N.E.Davidson, S.H.Kaufmann, S.B.Baylin, The estrogen receptor CpG island is methylated in most hematopoietic neoplasms, Cancer Res. 56 (1996) 973-977.
[84] J.P.Issa, B.A.Zehnbauer, S.H.Kaufmann, M.A.Biel, S.B.Baylin, HIC1 hypermethylation is a late event in hematopoietic neoplasms, Cancer Res. 57 (1997) 1678-1681.
[85] P.G.Corn, B.D.Smith, E.S.Ruckdeschel, D.Douglas, S.B.Baylin, J.G.Herman, E-cadherin expression is silenced by 5' CpG island methylation in acute leukemia, Clin.Cancer Res. 6 (2000) 4243-4248.
[86] S.Kawano, C.W.Miller, A.F.Gombart, C.R.Bartram, Y.Matsuo, H.Asou, A.Sakashita, J.Said, E.Tatsumi, H.P.Koeffler, Loss of p73 gene expression in leukemias/lymphomas due to hypermethylation, Blood 94 (1999) 1113-1120.
[87] M.Liu, T.Taketani, R.Li, J.Takita, T.Taki, H.W.Yang, H.Kawaguchi, K.Ida, Y.Matsuo, Y.Hayashi, Loss of p73 gene expression in lymphoid leukemia cell lines is associated with hypermethylation, Leuk.Res. 25 (2001) 441-447.
[88] J.Roman-Gomez, J.A.Castillejo, A.Jimenez, M.G.Gonzalez, F.Moreno, M.C.Rodriguez, M.Barrios, J.Maldonado, A.Torres, 5' CpG island hypermethylation is associated with transcriptional silencing of the p21(CIP1/WAF1/SDI1) gene and confers poor prognosis in acute lymphoblastic leukemia, Blood 99 (2002) 2291-2296.
[89] O.E.Bechter, W.Eisterer, M.Dlaska, T.Kuhr, J.Thaler, CpG island methylation of the hTERT promoter is associated with lower telomerase activity in B-cell lymphocytic leukemia, Exp.Hematol. 30 (2002) 26-33.
[90] P.E.Crossen, M.J.Morrison, Hypermethylation of the M27beta (DXS255) locus in chronic B-cell leukaemia, Br.J.Haematol. 100 (1998) 191-193.
[91] J.R.Melki, P.C.Vincent, R.D.Brown, S.J.Clark, Hypermethylation of E-cadherin in leukemia, Blood 95 (2000) 3208-3213.
[92] M.Esteller, G.Gaidano, S.N.Goodman, V.Zagonel, D.Capello, B.Botto, D.Rossi, A.Gloghini, U.Vitolo, A.Carbone, S.B.Baylin, J.G.Herman, Hypermethylation of the DNA repair gene O(6)-methylguanine DNA methyltransferase and survival of patients with diffuse large B-cell lymphoma, J.Natl.Cancer Inst. 94 (2002) 26-32.
[93] L.L.P.Siu, J.K.Cheung Chan, K.F.Wong, Y.L.Kwong, Specific Patterns of Gene Methylation in Natural Killer Cell Lymphomas : p73 Is Consistently Involved, Am.J.Pathol. 160 (2002) 59-66.
[94] R.A.Katzenellenbogen, S.B.Baylin, J.G.Herman, Hypermethylation of the DAP-kinase CpG island is a common alteration in B-cell malignancies, Blood 93 (1999) 4347-4353.
[95] A.S.Baur, P.Shaw, N.Burri, F.Delacretaz, F.T.Bosman, P.Chaubert, Frequent methylation silencing of p15(INK4b) (MTS2) and p16(INK4a) (MTS1) in B-cell and T-cell lymphomas, Blood 94 (1999) 1773-1781.
[96] J.P.Issa, B.A.Zehnbauer, C.I.Civin, M.I.Collector, S.J.Sharkis, N.E.Davidson, S.H.Kaufmann, S.B.Baylin, The estrogen receptor CpG island is methylated in most hematopoietic neoplasms, Cancer Res. 56 (1996) 973-977.
[97] J.P.Issa, B.A.Zehnbauer, S.H.Kaufmann, M.A.Biel, S.B.Baylin, HIC1 hypermethylation is a late event in hematopoietic neoplasms, Cancer Res. 57 (1997) 1678-1681.
[98] J.M.Taylor, P.H.Kay, D.V.Spagnolo, The diagnositc significance of Myf-3 hypermethylation in malignant lymphoproliferative disorders, Leukemia 15 (2001) 583-589.
[99] M.V.Mateos, R.Garcia-Sanz, R.Lopez-Perez, A.Balanzategui, M.I.Gonzalez, J.Fernandez-Calvo, M.J.Moro, J.Hernandez, M.D.Caballero, M.Gonzalez, J.F.San Miguel, p16/INK4a gene inactivation by hypermethylation is associated with aggressive variants of monoclonal gammopathies, Hematol.J. 2 (2001) 146-149.
[100] G.Guillerm, E.Gyan, D.Wolowiec, T.Facon, H.Avet-Loiseau, K.Kuliczkowski, F.Bauters, P.Fenaux, B.Quesnel, p16(INK4a) and p15(INK4b) gene methylations in plasma cells from monoclonal gammopathy of undetermined significance, Blood 98 (2001) 244-246.
[101] T.Uchida, T.Kinoshita, T.Ohno, H.Ohashi, H.Nagai, H.Saito, Hypermethylation of p16INK4A gene promoter during the progression of plasma cell dyscrasia, Leukemia 15 (2001) 157-165.
[102] G.Guillerm, E.Gyan, D.Wolowiec, T.Facon, H.Avet-Loiseau, K.Kuliczkowski, F.Bauters, P.Fenaux, B.Quesnel, p16(INK4a) and p15(INK4b) gene methylations in plasma cells from monoclonal gammopathy of undetermined significance, Blood 98 (2001) 244-246.
[103] M.H.Ng, K.W.To, K.W.Lo, S.Chan, K.S.Tsang, S.H.Cheng, H.K.Ng, Frequent death-associated protein kinase promoter hypermethylation in multiple myeloma, Clin.Cancer Res. 7 (2001) 1724-1729.
[104] W.K.Hofmann, S.Takeuchi, M.A.Frantzen, D.Hoelzer, H.P.Koeffler, Loss of genomic imprinting of insulin-like growth factor 2 is strongly associated with cellular proliferation in normal hematopoietic cells, Exp.Hematol. 30 (2002) 318-323.
[105] G.S.Randhawa, H.Cui, J.A.Barletta, L.Z.Strichman-Almashanu, M.Talpaz, H.Kantarjian, A.B.Deisseroth, R.C.Champlin, A.P.Feinberg, Loss of imprinting in disease progression in chronic myelogenous leukemia, Blood 91 (1998) 3144-3147.
[106] H.K.Wu, R.Weksberg, M.D.Minden, J.A.Squire, Loss of imprinting of human insulin-like growth factor II gene, IGF2, in acute myeloid leukemia, Biochem.Biophys.Res.Commun. 231 (1997) 466-472.
[107] I.M.Morison, M.R.Eccles, A.E.Reeve, Imprinting of insulin-like growth factor 2 is modulated during hematopoiesis, Blood 96 (2000) 3023-3028.
[108] W.K.Hofmann, S.Takeuchi, M.A.Frantzen, D.Hoelzer, H.P.Koeffler, Loss of genomic imprinting of insulin-like growth factor 2 is strongly associated with cellular proliferation in normal hematopoietic cells, Exp.Hematol. 30 (2002) 318-323.
[109] H.Nakagawa, R.B.Chadwick, P.Peltomaki, C.Plass, Y.Nakamura, C.A.de la, Loss of imprinting of the insulin-like growth factor II gene occurs by biallelic methylation in a core region of H19-associated CTCF-binding sites in colorectal cancer, Proc.Natl.Acad.Sci.U.S.A 98 (2001) 591-596.
[110] D.Takai, F.A.Gonzales, Y.C.Tsai, M.J.Thayer, P.A.Jones, Large scale mapping of methylcytosines in CTCF-binding sites in the human H19 promoter and aberrant hypomethylation in human bladder cancer, Hum.Mol.Genet. 10 (2001) 2619-2626.
[111] S.J.Kuerbitz, J.Pahys, A.Wilson, N.Compitello, T.A.Gray, Hypermethylation of the imprinted NNAT locus occurs frequently in pediatric acute leukemia, Carcinogenesis 23 (2002) 559-564.
[112] C.Plass, P.D.Soloway, DNA methylation, imprinting and cancer, Eur.J.Hum.Genet. 10 (2002) 6-16.
[113] M.R.Yuille, A.Condie, E.M.Stone, J.Wilsher, P.S.Bradshaw, L.Brooks, D.Catovsky, TCL1 is activated by chromosomal rearrangement or by hypomethylation, Genes Chromosomes.Cancer 30 (2001) 336-341.
[114] P.M.Watt, R.Kumar, U.R.Kees, Promoter demethylation accompanies reactivation of the HOX11 proto-oncogene in leukemia, Genes Chromosomes.Cancer 29 (2000) 371-377.
[115] P.Kantharidis, A.El Osta, M.deSilva, D.M.Wall, X.F.Hu, A.Slater, G.Nadalin, J.D.Parkin, J.R.Zalcberg, Altered methylation of the human MDR1 promoter is associated with acquired multidrug resistance, Clin.Cancer Res. 3 (1997) 2025-2032.
[116] V.Lipsanen, P.Leinonen, L.Alhonen, J.Janne, Hypomethylation of ornithine decarboxylase gene and erb-A1 oncogene in human chronic lymphatic leukemia, Blood 72 (1988) 2042-2044.
[117] V.Lipsanen, P.Leinonen, L.Alhonen, J.Janne, Hypomethylation of ornithine decarboxylase gene and erb-A1 oncogene in human chronic lymphatic leukemia, Blood 72 (1988) 2042-2044.
[118] M.Hanada, D.Delia, A.Aiello, E.Stadtmauer, J.C.Reed, bcl-2 gene hypomethylation and high-level expression in B-cell chronic lymphocytic leukemia, Blood 82 (1993) 1820-1828.
[119] M.A.Gama-Sosa, V.A.Slagel, R.W.Trewyn, R.Oxenhandler, K.C.Kuo, C.W.Gehrke, M.Ehrlich, The 5-methylcytosine content of DNA from human tumors, Nucleic Acids Res. 11 (1983) 6883-6894.
[120] G.Qu, L.Dubeau, A.Narayan, M.C.Yu, M.Ehrlich, Satellite DNA hypomethylation vs. overall genomic hypomethylation in ovarian epithelial tumors of different malignant potential, Mutat.Res. 423 (1999) 91-101.
[121] J.Wahlfors, H.Hiltunen, K.Heinonen, E.Hamalainen, L.Alhonen, J.Janne, Genomic hypomethylation in human chronic lymphocytic leukemia, Blood 80 (1992) 2074-2080.
[122] M.Lubbert, DNA methylation inhibitors in the treatment of leukemias, myelodysplastic syndromes and hemoglobinopathies: clinical results and possible mechanisms of action, Curr.Top.Microbiol.Immunol. 249 (2000) 135-164.
[123] P.Wijermans, M.Lubbert, G.Verhoef, A.Bosly, C.Ravoet, M.Andre, A.Ferrant, Low-dose 5-aza-2'-deoxycytidine, a DNA hypomethylating agent, for the treatment of high-risk myelodysplastic syndrome: a multicenter phase II study in elderly patients, J.Clin.Oncol. 18 (2000) 956-962.
[124] M.Lubbert, P.Wijermans, R.Kunzmann, G.Verhoef, A.Bosly, C.Ravoet, M.Andre, A.Ferrant, Cytogenetic responses in high-risk myelodysplastic syndrome following low-dose treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine, Br.J.Haematol. 114 (2001) 349-357.
[125] R.L.Piekarz, R.Robey, V.Sandor, S.Bakke, W.H.Wilson, L.Dahmoush, D.M.Kingma, M.L.Turner, R.Altemus, S.E.Bates, Inhibitor of histone deacetylation, depsipeptide (FR901228), in the treatment of peripheral and cutaneous T-cell lymphoma: a case report, Blood 98 (2001) 2865-2868.
[126] H.Suzuki, E.Gabrielson, W.Chen, R.Anbazhagan, M.Van Engeland, M.P.Weijenberg, J.G.Herman, S.B.Baylin, A genomic screen for genes upregulated by demethylation and histone deacetylase inhibition in human colorectal cancer, Nat.Genet. (2002).
[127] Z.Zhang, Technology evaluation: MG-98, MethylGene, Curr.Opin.Mol.Ther. 3 (2001) 599-604.
[128] J.G.Herman, C.I.Civin, J.P.Issa, M.I.Collector, S.J.Sharkis, S.B.Baylin, Distinct patterns of inactivation of p15INK4B and p16INK4A characterize the major types of hematological malignancies, Cancer Res. 57 (1997) 837-841.
[129] J.F.Costello, M.C.Fruhwald, D.J.Smiraglia, L.J.Rush, G.P.Robertson, X.Gao, F.A.Wright, J.D.Feramisco, P.Peltomaki, J.C.Lang, D.E.Schuller, L.Yu, C.D.Bloomfield, M.A.Caligiuri, A.Yates, R.Nishikawa, H.H.Su, N.J.Petrelli, X.Zhang, M.S.O'Dorisio, W.A.Held, W.K.Cavenee, C.Plass, Aberrant CpG-island methylation has non-random and tumour-type-specific patterns, Nat.Genet. 24 (2000) 132-138.
[130] R.S.Gitan, H.Shi, C.M.Chen, P.S.Yan, T.H.Huang, Methylation-specific oligonucleotide microarray: a new potential for high-throughput methylation analysis, Genome Res. 12 (2002) 158-164.

Figure 1. Biological processes that could be subject to disruption by aberrant methylation during lineage development of a multipotent stem cell. At the top of the arrow are processes that could be influenced by hypomethylation events. At the bottom are processes that could be influenced by hypermethylation events.

Figure 2.Potential outcomes after treatment of leukemia with a demethylating agent. At the left is a schematic representation of a leukemic bone marrow. The large cells represent leukemic blasts; the small cells represent normal marrow elements. For the promoter of interest, “U” represents an unmethylated promoter; “M” represents a methylated promoter. Following treatment with a demethylating agent, various outcomes are depicted. The expected results of a methylation-sensitive PCR reaction (MS-PCR) for promoter methylation are shown in cartoon form as the presence or absence of a band on a gel.

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