Comparison of bisulfite modification of 5-methyldeoxycytidine and deoxycytidine residues

Wang, Richard Y.-H.; Gehrke, Charles W.; Ehrlich, Melanie

doi:10.1093/nar/8.20.4777

Cited by 218 publications

(134 citation statements)

References 23 publications

(35 reference statements)

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“…Previous studies reported p15 gene methylation in 18-60% of ALL patients (Wang et al, 1980;Iravani et al, 1997;Chim et al, 2001;GarciaManero et al, 2002b;Chim et al, 2003;Garcia-Manero et al, 2003;Roman-Gomez et al, 2004;Scott et al, 2004;Canalli et al, 2005;Roman-Gomez et al, 2005;Takeuchi et al, 2012). In contrast to these studies, Chen and Wu (2002) reported methylation in 90% of ALL patients, while Matsushita et al (2004) reported absence of p15 methylation.…”

Section: Discussionmentioning

confidence: 92%

DNA Hypermethylation of Cell Cycle (p15 and p16) and Apoptotic (p14, p53, DAPK and TMS1) Genes in Peripheral Blood of Leukemia Patients

Bodoor

Haddad

Alkhateeb

et al. 2014

Asian Pacific Journal of Cancer Prevention

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Aberrant DNA methylation of tumor suppressor genes has been reported in all major types of leukemia with potential involvement in the inactivation of regulatory cell cycle and apoptosis genes. However, most of the previous reports did not show the extent of concurrent methylation of multiple genes in the four leukemia types. Here, we analyzed six key genes (p14, p15, p16, p53, DAPK and TMS1) for DNA methylation using methylation specific PCR to analyze peripheral blood of 78 leukemia patients (24 CML, 25 CLL, 12 AML, and 17 ALL) and 24 healthy volunteers. In CML, methylation was detected for p15 (11%), p16 (9%), p53 (23%) and DAPK (23%), in CLL, p14 (25%), p15 (19%), p16 (12%), p53 (17%) and DAPK (36%), in AML, p14 (8%), p15 (45%), p53 (9%) and DAPK (17%) and in ALL, p15 (14%), p16 (8%), and p53 (8%). This study highlighted an essential role of DAPK methylation in chronic leukemia in contrast to p15 methylation in the acute cases, whereas TMS1 hypermethylation was absent in all cases. Furthermore, hypermethylation of multiple genes per patient was observed, with obvious selectiveness in the 9p21 chromosomal region genes (p14, p15 and p16). Interestingly, methylation of p15 increased the risk of methylation in p53, and vice versa, by five folds (p=0.03) indicating possible synergistic epigenetic disruption of different phases of the cell cycle or between the cell cycle and apoptosis. The investigation of multiple relationships between methylated genes might shed light on tumor specific inactivation of the cell cycle and apoptotic pathways.

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Section: Discussionmentioning

confidence: 92%

DNA Hypermethylation of Cell Cycle (p15 and p16) and Apoptotic (p14, p53, DAPK and TMS1) Genes in Peripheral Blood of Leukemia Patients

Bodoor

Haddad

Alkhateeb

et al. 2014

Asian Pacific Journal of Cancer Prevention

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“…25 For bisulfite treatment, 1 g of genomic DNA was denatured by NaOH and modified by sodium bisulfite, which converts all unmethylated cytosines to uracil while methylated cytosines remain unchanged. 26 The modified DNA was purified using a Wizard DNA purification kit (Promega, Madison, WI), treated with NaOH to desulfonate, precipitated with ethanol, and resuspended in water.…”

Section: Dna Extraction and Bisulfite Treatmentmentioning

confidence: 99%

Establishment and Validation of Real-Time Polymerase Chain Reaction Method for CDH1 Promoter Methylation

Toyooka

Maitra

et al. 2002

The American Journal of Pathology

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Aberrant methylation of the promoter region has emerged as the major mechanism for silencing tumor suppressor genes. However, for some genes, such as E-cadherin (CDH1), methylation and protein expression demonstrate considerable heterogeneity, making correlations difficult. We compared methylation and protein expression status of CDH1 in 56 primary breast carcinomas using semiquantitative assays. Aberrant CDH1 methylation was studied by methylation-specific polymerase chain reaction (MSP) and semiquantitative real-time MSP assays. The Cdh1 expression was investigated by immunostaining on archival formalin-fixed sections from 34 primary carcinomas and their accompanying normal epithelium and preinvasive and metastatic lesions. Membrane-specific Cdh1 expression in the neoplastic cells was quantified by image analysis using an automated cellular imaging system and a continuous score. Aberrant promoter methylation of the CDH1 was present in 24 of 56 (43%) breast carcinomas by MSP assay. There was excellent concordance between the standard MSP assay and the real-time assay (91%, P < 0.0001). The concordance between loss of Cdh1 expression and CDH1 methylation by standard MSP was 71% (P ‫؍‬ 0.02). Furthermore, there was a strong correlation between the semiquantitative assays for methylation and protein expression (r ‫؍‬ 0.47, P ‫؍‬ 0.005). We conclude that promoter methylation of CDH1 significantly correlated with the Cdh1 expression level, demonstrating that epigenetic silencing is a valid pathway for silencing of tumor suppressor genes in primary breast carcinomas.

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“…Treatment with sodium bisulfite can be used to convert methylated and unmethylated DNA to different sequences [9,10] (Fig. 1a).…”

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

Parallel assessment of CpG methylation by two-color hybridization with oligonucleotide arrays

Balog

Souza

Tang

et al. 2002

Analytical Biochemistry

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We have developed a method for the parallel analysis of multiple CpG sites in genomic DNA for their state of methylation. Hypermethylation of CpG islands within the promoters and 5′ exons of genes has been found to be a mechanism of transcriptional inactivation associated with a variety of tumors. The method that we developed relies on the differential reactivity of methylated and unmethylated cytosines with sodium bisulfite, which exclusively converts unmethylated cytosines to deoxyuracils. The resulting sequence changes are determined with single-nucleotide resolution by hybridization to an oligonucleotide array. Cohybridization with a reference sample containing a different label provides an internal standard for assessment of methylation state. This method provides advantages in parallelism over existing methods of methylation analysis. We have demonstrated this technique with a region from the promoter of the tumor suppressor gene p16, which is hypermethylated in many cancers. KeywordsHypermethylation; CpG island; Oligonucleotide array; Sodium bisulfite; Tumor suppressor Methylation of cytosines in CpG dinucleotides is an important mechanism of transcriptional regulation. It is involved in a diversity of normal biological processes such as X chromosome inactivation [1] and transcriptional regulation of imprinted genes [2]. Aberrant methylation of cytosines can also effect transcriptional inactivation of certain tumor suppressor genes, associated with a number of human cancers [3,4]. Cytosine methylation in CpG-rich areas (CpG islands) located in the promoter regions of some genes is of special regulatory importance. Therefore, wide-scope mapping of methylation sites in CpG islands is important for understanding both normal and pathological cellular processes. Furthermore, methylation of certain sites might serve as an important marker for early diagnosis and treatment decisions of some cancers [5,6]. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptA variety of methods have been used to identify sites of DNA methylation. One common method has relied on the inability of restriction endonucleases to cleave sequences that contain one or more methylated cytosines [7]. Genomic DNA is fragmented with methylation-sensitive restriction enzymes, and cleavage at the site of interest is probed by Southern blotting, PCR, or hybridization to a CpG island array [8]. This method is limited to sites that fall within the recognition sequences of methylation-sensitive restriction enzymes.Alternative methods rely on the differential chemical reactivities of cytosine and 5-methyl cytosine with reagents such as sodium bisulfite, hydrazine, or permanganate. Treatment with sodium bisulfite can be used to convert methylated and unmethylated DNA to different sequences [9,10] (Fig. 1a). Under appropriate conditions, unmethylated cytosines in DNA react with sodium bisulfite to yield deoxyuracil, which behaves as thymidine in enzymatic template-directed polymerization. Methylated cytosines, however, ar...

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Comparison of bisulfite modification of 5-methyldeoxycytidine and deoxycytidine residues

Cited by 218 publications

References 23 publications

DNA Hypermethylation of Cell Cycle (p15 and p16) and Apoptotic (p14, p53, DAPK and TMS1) Genes in Peripheral Blood of Leukemia Patients

DNA Hypermethylation of Cell Cycle (p15 and p16) and Apoptotic (p14, p53, DAPK and TMS1) Genes in Peripheral Blood of Leukemia Patients

Establishment and Validation of Real-Time Polymerase Chain Reaction Method for CDH1 Promoter Methylation

Parallel assessment of CpG methylation by two-color hybridization with oligonucleotide arrays

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