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Prepublished online as a Blood First Edition Paper on May 13, 2002; DOI 10.1182/blood-2001-11-0026.
NEOPLASIA
From the First Department of Internal Medicine, and the
First Department of Pathology, Nagoya University School of Medicine;
the Department of Hematology, Nagoya National Hospital; and the Aichi
Blood Disease Research Foundation, Nagoya, Japan; and from
the Department of Pediatrics, School of Medicine, Hirosaki University,
Japan.
The cyclin-dependent kinase inhibitor
p57KIP2 is thought to be a
potential tumor suppressor gene (TSG). The present study examines this
possibility. We found that the expression of
p57KIP2 gene is absent in various
hematological cell lines. Exposing cell lines to the DNA demethylating
agent 5-aza-2'-deoxycytidine restored
p57KIP2 gene expression. Bisulfite
sequencing analysis of its promoter region showed that
p57KIP2 DNA was completely methylated in
cell lines that did not express the
p57KIP2 gene. Thus, DNA methylation of
its promoter might lead to inactivation of the
p57KIP2 gene. DNA methylation of this
region is thought to be an aberrant alteration, since DNA was not
methylated in normal peripheral blood mononuclear cells or in
reactive lymphadenitis. Methylation-specific polymerase chain
reaction analysis found frequent DNA methylation of the
p57KIP2 gene in primary diffuse large
B-cell lymphoma (54.9%) and in follicular lymphoma (44.0%), but
methylation was infrequent in myelodysplastic syndrome and adult T-cell
leukemia (3.0% and 2.0%, respectively). These findings directly
indicate that the profile of the p57KIP2
gene corresponds to that of a TSG.
(Blood. 2002;100:2572-2577) Cell cycle progression is promoted by cyclins and
cyclin-dependent kinases (CDKs) and is counterbalanced by CDK
inhibitors.1 These components are regulated in the normal
cell cycle, and disrupted regulation is closely related to
tumorigenesis. For example, activation of cyclin D1 by bcl-1
translocation (t(11;14)(q13;q23)) is a critical genetic alteration in
mantle cell lymphoma,2,3 and the CDK inhibitor
p16INK4A is genetically lost or mutated
in many malignancies.4 CDK inhibitors have been
categorized into 2 families. One family, inhibitors of CDK4
(INK4s), includes p16 and p15, has repeated ankyrinlike
sequences, and is a specific inhibitor of CDK4 and CDK6, resulting in competing cyclin D.5,6
The other family, kinase inhibitor proteins (KIPs), includes
p21CIP1, p27KIP1, and
p57KIP2,7-10 and this family potently
inhibits the binding of several cyclin/CDK complexes. The presence of
mutations and deletions in INK4s indicates that they are tumor
suppressor genes (TSGs)4 and that their function as
negative regulators of cell proliferation fits the profile of TSGs.
Because the KIP family also consists of CDK inhibitors, genetic
alterations of the p21CIP1
gene11 and p57KIP2
genes12-14 have been extensively studied. In particular,
the p57KIP2 gene is located at chromosome
11p15.5, a region implicated in sporadic cancers, including those of
the breast,15 liver,16 and
bladder.17 Beckwith-Wiedemann syndrome
(BWS),18 which is characterized by the somatic overgrowth
of various tissues, including the kidneys, liver, and skeletal muscle,
and by a predisposition to embryonal tumors of these organs (Wilms
tumor, hepatoblastoma, and embryonal rhabdomyosarcoma), is linked
to this region.19 Although germ line point mutations of
the p57KIP2 gene resulting in protein
truncation have been identified in some patients with
BWS,20,21 somatic mutations have not been found in
sporadic cancers, including Wilms tumor.12-14,21 The p57KIP2 gene is also an imprinted gene
that preferentially expresses the maternal allele. In mice, genomic
imprinting of the p57KIP2 gene is
absolute and is regulated by DNA methylation,22 but DNA
methylation has not been detected in healthy
humans.23,24 The maternal monoallelic expression of the
p57KIP2 gene might be regulated by a
mechanism other than DNA methylation in humans.
DNA methylation of CpG islands in the promoter region leads to
epigenetic gene inactivation by transcriptional silencing. Recently,
aberrant DNA methylation of candidate TSGs have been found in various
tumors,25-27 and this mechanism might be an alternative pathway of TSG inactivation. DNA hypermethylation of the INK4 family is
frequent in lymphoid malignancies,25,26 and this mechanism
is responsible for the inactivation of these genes.
Here we demonstrate that the aberrant DNA methylation of the
promoter region of p57KIP2 gene is found
in hematological malignancies, and we examine the relationship between
DNA methylation of the p57KIP2 gene and
its gene expression.
Samples and DNA/RNA preparation
Reverse transcriptase-polymerase chain reaction
Culture with 5-aza-2'-deoxycytidine
Bisulfite sequencing We modified 500 ng genomic DNA extract from clinical samples and cell lines with sodium bisulfite as described26 using a DNA modification kit (CpGenome DNA Modification Kit; Oncor, Gaithersburg, MD) according to the manufacturer's instructions. Modified DNA (25 ng) was amplified by nested PCR to obtain the promoter sequence of the p57KIP2 gene. First-round PCR consisted of 35 cycles at an annealing temperature of 56°C, with the use of the primers MS1 (5'-TTTCGTTTGTAGATAAAGGA-3') and MS2 (5'-CTAACTATCCGATAATAAACTCTTCTA-3') (Figure 1). With the use of one twentieth of the first-round PCR product as a template, second-round PCR consisted of 35 cycles at an annealing temperature of 60°C with the primers MS3 (5'-GGGGGTGGGGAGTGTTGT-3') and MS4 (5'-ATATTTTCAATTTCAACAACACCA-3') (Figure 1). Under these conditions, we could assess the DNA methylation status of p57KIP2 gene at the position 400 to 149 bp upstream of the transcription initiation site. PCR products were separated on 2% low-melting agarose gels, excised, and then digested with -agarase (New England Biolabs, Beverly, MA). The digestion
products were subcloned into the PGEM-Teasy vector, and at least 10 clones of each product were subjected to cycle sequencing (PE Applied
Biosystems, Warrington, United Kingdom) and were analyzed by means of
an ABI 310 (Applied Biosystems, Foster City, CA).
Methylation-specific PCR (MSP) DNA modified with sodium bisulfite was selectively amplified by PCR with the use of primers MS1 and MS2 under the conditions described above. The concentrated modified DNA was then analyzed by MSP with the use of primer sets specific for unmethylated DNA (MSP-U, 5'-TTTGTTTTGTGGTTGTTAATTAGTTGT-3', and MSP-A, 5'-ACACAACGCACTTAACCTATAA-3') and methylated DNA (MSP-M, 5'-CGCGGTCGTTAATTAGTCGC-3', and MSP-A). The annealing temperatures were 56°C for MSP-U with MSP-A, and 63°C for MSP-M with MSP-A. Each PCR was "hot-started," and the products were separated on 2% agarose gels and then visualized by ethidium bromide staining.The sensitivity of the MSP method was confirmed with the use of a mixture of bisulfite-modified DNA of HL60 and of U937, which have completely methylated and unmethylated p57KIP2 gene promoter sequences, respectively, at ratios of 100:0, 10:90, 1:99, 0.1:99.9, and 0:100. PCR-SSCP Mutations of the p57KIP2 gene were screened by PCR-SSCP analysis29 with the use of primers as listed in a previous report.12 Thirty cycles of PCR amplification proceeded at annealing temperatures ranging from 56°C to 62°C in the presence of 1 µCi (0.037 MBq) -[32P]-deoxycytidine triphosphate
(-32P]-dCTP) per reaction. The
products were separated on 0.5 × MDE gels (FMC BioProducts,
Rockland, ME). Bands were exposed on phosphorimaging plates and
evaluated by means of a laser image analyzer (FUJI, Kanagawa, Japan).
Expression of p57KIP2 gene We examined p57KIP2 gene expression in 17 hematological cell lines, 2 lymphadenitis samples, and 1 normal peripheral blood mononuclear cell (PBMNC) sample by RT-PCR using the primer set KIP2-1 and KIP2-2 (Figure 1). Expression of the p57KIP2 gene was undetectable in 5 cell lines (RAMOS, DAUDI, RAJI, HS-SULTAN, and HL60) after 35 amplification cycles (Figure 2). These cell lines were derived from diverse hematological malignancies, but loss of expression tended to be more frequent in lymphoid cell lines of the B-cell lineage. PCR analysis of genomic DNA using primers for PCR-SSCP analysis showed that none of the cell lines analyzed here had genetically lost the p57KIP2 gene (data not shown).
Restoration of p57KIP2 gene expression by 5-aza-2'-deoxycytidine We examined the mechanism of down-regulated p57KIP2 gene expression. When 2 cell lines (HL60 and RAJI) that did not express p57KIP2 gene were exposed to the demethylating agent 5-aza-2'-deoxycytidine (0, 5, or 10 µM), p57KIP2 gene expression was restored in both (Figure 3). The unmethylated status at the promotor region of the p57KIP2 gene in both cell lines was detected after this procedure. Methylated bands remained after exposure to 5-aza-2'-deoxycytidine in RAJI cells, but the methylated bands disappeared from HL60 cells. All methylated sequences of the p57KIP2 gene in HL60 might have become unmethylated by this procedure. In RAJI cells, this conversion might not have been complete. These data suggested that DNA methylation of the p57KIP2 gene or of genes that transcriptionally regulate p57KIP2 might be one mechanism of inactivation of the p57KIP2 gene expression.
Bisulfite sequence of the p57KIP2 gene promoter region We further examined the involvement of DNA methylation in relation to p57KIP2 gene expression. We performed bisulfite genomic sequencing of 46 CpG sites at the promoter region of the p57KIP2 gene (Figure 1) in 2 samples of lymphadenitis, 1 PBMNC sample from a healthy volunteer, 2 cell lines that expressed p57KIP2 gene (NAMALWA and THP-6), and 4 cell lines that did not (RAMOS, RAJI, HL60, and HS-SULTAN; Figure 4). The sequence analyzed here contained 12 putative transcriptional factor-binding sites.12 The lymphadenitis samples and normal PBMNC sample had no DNA methylation in 46 CpG sites of the promoter region. The presence of p57KIP2 gene expression was confirmed in these samples by RT-PCR (Figure 2). NAMALWA expressed the p57KIP2 gene, and the sequence in this region was completely unmethylated. THP-6 also expressed the p57KIP2 gene. These sequences were methylated in 6 of 12 clones sequenced, and unmethylated in another 6. Unmethylated sequences at this region might be responsible for expression of the p57KIP2 gene. All of the sequences were almost completely methylated in 3 of 4 cell lines without p57KIP2 gene expression, which was consistent with the loss of this gene expression. Although RAMOS did not express the p57KIP2 gene, its promoter region was completely unmethylated. Thus, silencing mechanisms other than DNA methylation in the promoter region might exist. The completely unmethylated p57KIP2 gene promoter in nonmalignant samples suggested that p57KIP2 promoter DNA methylation itself is aberrant in healthy individuals.
Methylation of p57KIP2 gene detected by MSP We screened for DNA methylation of the promoter region of the p57KIP2 gene, using the MSP method. We initially examined the sensitivity of the method using mixed samples of completely unmethylated and methylated cell lines. The results showed that the MSP method could detect one methylated cell among 1 × 102 unmethylated cells (Figure 5). We then examined mRNA expression of the p57KIP2 gene in 17 hematological cell lines and compared the results of MSP analysis and bisulfite sequencing. The results of the 2 methods to detect DNA methylation were comparable (Figures 2 and 4). DAUDI cells have an unmethylated band, but mRNA was not detected. This discrepancy was also revealed in the RAMOS cell line by bisulfite sequencing, indicating an alternative mechanism of silencing the p57KIP2 gene. Six samples of lymphadenitis and 7 of normal PBMNCs were analyzed by MSP, and methylated bands were undetectable (Figure 2 and 6). This finding means that nonmalignant mononuclear cells from peripheral blood and lymphadenitis tissue do not have a methylated promoter region in the p57KIP2 gene. We then screened the methylation status of the p57KIP2 gene promoter in primary hematological malignancies, including 71 DLBCL, 18 FL, 18 CLL, 26 MM, 6 ALL, 53 ATL, 14 AML, and 52 MDS samples, using the MSP method. The p57KIP2 gene promoter was methylated in 39 of 71 DLBCL samples (54.9%), in 4 of 26 MM samples (15.3%), and in 1 of 51 ATL samples (2.0%; Figure 7). Table 1 lists the frequencies of p57KIP2 gene methylation in various hematological malignancies. These data suggested that aberrant DNA methylation of p57KIP2 gene is detected in some populations of hematological malignancies, and that the frequency of this methylation is higher in lymphoid malignancies of the B-cell phenotype.
Mutation of coding region of p57KIP2 gene We screened for mutations of the p57KIP2 gene coding region in 31 of these 71 DLBCL samples by PCR-SSCP analysis. We did not find any mutations of the p57KIP2 gene (data not shown).
The present study indicates that aberrant DNA methylation of p57KIP2 gene at its promoter region might constitute one mechanism of silencing p57KIP2 gene expression. We also found this epigenetic alteration not only in cell lines but also in primary hematological malignancies. In addition to point mutations or gene deletions, transcriptional repression by the hypermethylation of promoter sequences may be an alternative mechanism for TSG inactivation in cancers. Hypermethylation of CpG islands of TSG was originally detected in the retinoblastoma gene.30 The methylation of CpG sites in the p16INK4A gene has recently been recognized as a mechanism that suppresses its transcription in lymphoma, myeloma, and solid tumors.26,31,32 The major biological effect of p16INK4A is to halt cell cycle progression at the G1/S boundary, and the loss of p16INK4A function may result in cancer progression by allowing unregulated cellular proliferation. Because of its function as a CDK inhibitor like p16INK4A and p15INK4B, and its chromosomal location (11p15.5, which is recurrently deleted in various tumors), the p57KIP2 gene has been considered a good TSG candidate. Nevertheless, no direct evidence has confirmed this notion. Expression of the p57KIP2 gene is decreased in gastric cancer,33 bladder cancer,34 blastic crisis of chronic myelogenous leukemia,35 and Wilms tumor.24,36 These reports weakly supported one aspect of p57KIP2 as a TSG candidate. The p57KIP2 gene is imprinted, which means preferential maternal expression.37 The mechanism of p57KIP2 gene imprinting is now under investigation. So far, no evidence correlates the expression of p57KIP2 and DNA methylation at the promoter region under physiological conditions. In addition, we did not detect DNA methylation of the p57KIP2 gene in the promoter region in normal blood or lymphadenitis samples. Methylation at the promoter region might not contribute to the monoallelic expression of this gene. However, the DNA might be methylated at a site other than at the promoter region, resulting in p57KIP2 gene imprinting. The chromosomal 11p15.5 region harbors several imprinted genes, and clusters of these genes might be divided into 2 separate imprinted domains.13 The centromeric domain contains p57KIP2 and the telomeric domain contains H19 and IGF2, and these regions are independently regulated.13 In the centromeric region, a paternally expressed transcript, LIT1,38 located within KvLQT1 and transcribed in antisense orientation to it, might play a role in p57KIP2 regulation similar to that of H19 in IGF2 regulation.13 The establishment of an imprint of the p57KIP2 gene might be coordinately regulated throughout the entire imprinted domain. Shin et al33 showed promoter methylation of p57KIP2 gene by means of a conventional PCR-based method in gastric cancer. We did not detect p57KIP2 mRNA expression in several hematological cell lines by RT-PCR. Treatment of RAJI and HL60, which do not express the p57KIP2 gene, with the demethylating agent 5-aza-2'-deoxycytidine restored p57KIP2 gene expression. Sequence analysis of the p57KIP2 gene promoter region after bisulfite modification precisely revealed the methylation status. We found that cell lines with complete DNA methylation at promoter region of p57KIP2 gene did not express this gene. In contrast, cell lines that expressed the p57KIP2 gene had unmethylated promoter sequences. In THP-6, the p57KIP2 gene promoter was unmethylated in half of the clones analyzed by bisulfite sequencing and methylated in the other half. We could not determine which clones were of maternal origin, but these unmethylated promoter sequences might lead to expression of the p57KIP2 gene. RAMOS and DAUDI did not express the p57KIP2 gene despite the presence of unmethylated promoter sequence. There might exist mechanisms to inactivate the p57KIP2 gene mechanisms other than DNA methylation in the promoter region, such as DNA methylation of p57KIP2 gene at other regions, deregulation of a gene transcriptionally upstream of the p57KIP2 gene, or histone deacetylation. The formation of inactive chromatin through histone deacetylation is an alternative mechanism for inactivation of the p57KIP2 gene in gastric cancer cells.33 Bisulfite sequencing revealed no methylated sequences of the p57KIP2 promoter region in nonmalignant cells, and MSP analysis did not detect methylation of this region in 6 lymphadenitis samples and 7 normal PBMNC samples. These findings suggested that complete DNA methylation of the p57KIP2 gene promoter silenced this gene, and that methylation of this region itself was an aberrant alteration in lymphoid tissues. Thus, epigenetic silencing of the p57KIP2 gene revealed that this gene might be a TSG candidate despite the absence of genetic mutations. MSP is a simple and sensitive method of detecting DNA methylation and requires only small specimens containing limited amounts of DNA.26 We used this method to detect aberrant DNA methylation of the p57KIP2 gene promoter region at different frequencies among primary hematological malignancies. This region was 54.9% methylated in DLBCL, but 1.9% in ATL and 0% in AML. This observation suggested that the aberrant p57KIP2 gene methylation was not random among the hematological malignancies, and that the samples with the B-cell phenotype were preferentially affected. Such differences in the frequencies of methylation of TSGs among hematological malignancies have been identified in the p16INK4A and p15INK4B genes.39 An understanding of the relationship between methylation of the p57KIP2 gene and the clinical outcome of primary tumors is necessary to determine the clinical significance of such epigenetic alterations. Unfortunately, clinical information about the patients analyzed in this study is rather sparse, so we could not assess these points. To elucidate the role of aberrant DNA methylation of the p57KIP2 gene in primary hematological malignancies, the expression level of the p57KIP2 gene should be assessed in tumor specimens. RT-PCR detected p57KIP2 mRNA in primary tumors even when the p57KIP2 gene was methylated (data not shown). We considered these mRNAs to be expressed by normal background cells of tumor specimens. We are now attempting to determine the protein level of KIP2 by immunohistochemical means in primary tumors. Among several studies of p57KIP2-deficient mice,40,41 Zhang et al40 showed that a lack of kip2 causes some features of BWS. However, Takahashi et al41 reported that most BWS symptoms could not be reproduced. Both groups of investigators found that mice lacking kip2 did not develop tumors, in either the kidney or liver. These results do not substantiate the notion that the p57KIP2 gene is a TSG. However, these results were obtained from mice models and cannot be completely extrapolated to humans. Because human neoplasms are thought to develop through several steps, analyses of double-knockout mice that lack p57KIP2 and other candidate TSGs might be required to more precisely elucidate the effects of the p57KIP2 gene on tumor formation. The presence of genetic mutations is thought to be a hallmark of many TSGs and TSG candidates. Recent investigations into epigenetic alterations in TSGs have highlighted DNA methylation as a powerful mechanism of TSG inactivation. Our findings indicate that methylation of the promoter region is a pathway of p57KIP2 gene inactivation. We also found that the p57KIP2 gene is actually methylated in primary lymphoid malignancies as an aberrant and epigenetic event. This would support the notion that the p57KIP2 gene is a candidate TSG. Further study of its biological effect on tumorigenesis might further delineate the profile of p57KIP2 gene as a TSG.
We thank Ms K. Kondo for excellent technical assistance.
Submitted November 26, 2001; accepted April 27, 2002.
Prepublished online as Blood First Edition Paper, May 13, 2002; DOI 10.1182/blood-2001-11-0026.
Supported by Grants-in-Aid for Cancer Research (11S-1 and 11-8) from the Ministry of Health, Labor and Welfare, Japan.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Hirokazu Nagai, Department of Hematology, Nagoya National Hospital, 4-1-1 Sannomaru, Naka-ku, Nagoya, 460-0001, Japan; e-mail: nagaih{at}nnh.hosp.go.jp.
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© 2002 by The American Society of Hematology.
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H. Shi, J. Guo, D. J. Duff, F. Rahmatpanah, R. Chitima-Matsiga, M. Al-Kuhlani, K. H. Taylor, O. Sjahputera, M. Andreski, J. E. Wooldridge, et al. Discovery of novel epigenetic markers in non-Hodgkin's lymphoma Carcinogenesis, January 1, 2007; 28(1): 60 - 70. [Abstract] [Full Text] [PDF]
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 N Diaz-Meyer, Y Yang, S N Sait, E R Maher, and M J Higgins Alternative mechanisms associated with silencing of CDKN1C in Beckwith-Wiedemann syndrome J. Med. Genet., August 1, 2005; 42(8): 648 - 655. [Abstract] [Full Text] [PDF]
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 N. Sato, H. Matsubayashi, T. Abe, N. Fukushima, and M. Goggins Epigenetic Down-Regulation of CDKN1C/p57KIP2 in Pancreatic Ductal Neoplasms Identified by Gene Expression Profiling Clin. Cancer Res., July 1, 2005; 11(13): 4681 - 4688. [Abstract] [Full Text] [PDF]
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 C. Bueso-Ramos, Y. Xu, T. J. McDonnell, S. Brisbay, S. Pierce, H. Kantarjian, G. Rosner, and G. Garcia-Manero Protein Expression of a Triad of Frequently Methylated Genes, p73, p57Kip2, and p15, Has Prognostic Value in Adult Acute Lymphocytic Leukemia Independently of Its Methylation Status J. Clin. Oncol., June 10, 2005; 23(17): 3932 - 3939. [Abstract] [Full Text] [PDF]
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 C. Pantoja, L. de los Rios, A. Matheu, F. Antequera, and M. Serrano Inactivation of Imprinted Genes Induced by Cellular Stress and Tumorigenesis Cancer Res., January 1, 2005; 65(1): 26 - 33. [Abstract] [Full Text] [PDF]
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 J Liu, X-D Li, A Vaheri, and R Voutilainen DNA methylation affects cell proliferation, cortisol secretion and steroidogenic gene expression in human adrenocortical NCI-H295R cells J. Mol. Endocrinol., December 1, 2004; 33(3): 651 - 662. [Abstract] [Full Text] [PDF]
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 J. M. Scandura, P. Boccuni, J. Massague, and S. D. Nimer Transforming growth factor {beta}-induced cell cycle arrest of human hematopoietic cells requires p57KIP2 up-regulation PNAS, October 19, 2004; 101(42): 15231 - 15236. [Abstract] [Full Text] [PDF]
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T. Takahashi, N. Shivapurkar, E. Riquelme, H. Shigematsu, J. Reddy, M. Suzuki, K. Miyajima, X. Zhou, B. N. Bekele, A. F. Gazdar, et al. Aberrant Promoter Hypermethylation of Multiple Genes in Gallbladder Carcinoma and Chronic Cholecystitis Clin. Cancer Res., September 15, 2004; 10(18): 6126 - 6133. [Abstract] [Full Text] [PDF]
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 G. Li, J. Domenico, J. J. Lucas, and E. W. Gelfand Identification of Multiple Cell Cycle Regulatory Functions of p57Kip2 in Human T Lymphocytes J. Immunol., August 15, 2004; 173(4): 2383 - 2391. [Abstract] [Full Text] [PDF]
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N Diaz-Meyer, C D Day, K Khatod, E R Maher, W Cooper, W Reik, C Junien, G Graham, E Algar, V M Der Kaloustian, et al. Silencing of CDKN1C (p57KIP2) is associated with hypomethylation at KvDMR1 in Beckwith-Wiedemann syndrome J. Med. Genet., November 1, 2003; 40(11): 797 - 801. [Abstract] [Full Text] [PDF]
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 M. Du, L. G. Beatty, W. Zhou, J. Lew, C. Schoenherr, R. Weksberg, and P. D. Sadowski Insulator and silencer sequences in the imprinted region of human chromosome 11p15.5 Hum. Mol. Genet., August 1, 2003; 12(15): 1927 - 1939. [Abstract] [Full Text] [PDF]
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 L. Shen, M. Toyota, Y. Kondo, T. Obata, S. Daniel, S. Pierce, K. Imai, H. M. Kantarjian, J.-P. J. Issa, and G. Garcia-Manero Aberrant DNA methylation of p57KIP2 identifies a cell-cycle regulatory pathway with prognostic impact in adult acute lymphocytic leukemia Blood, May 15, 2003; 101(10): 4131 - 4136. [Abstract] [Full Text] [PDF]
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R. Weksberg, A. C. Smith, J. Squire, and P. Sadowski Beckwith-Wiedemann syndrome demonstrates a role for epigenetic control of normal development Hum. Mol. Genet., April 2, 2003; 12(90001): R61 - 68. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
282 to 
