Quantitative reversed-phase high-performance liquid chromatography of major and modified nucleosides in dna

Gehrke, Charles W.; McCune, Roy A.; Gama-Sosa, Miguel A.; Ehrlich, Melanie; Kuo, Kenneth C.

doi:10.1016/s0021-9673(01)89189-5

Cited by 200 publications

(121 citation statements)

References 26 publications

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“…The 5-methylcytosine content was quantified by chromatographic separation of five deoxyribonucleotides, 5-methyl-dCMP, dCMP, dAMP, dGMP and TMP, as reported. [19][20][21] Briefly, genomic DNA was degraded by treatment with DNase I and Nuclease P1. After filtration with a 0.45-µm filter, the sample was subjected to highpressure liquid chromatography (HPLC).…”

Section: Methodsmentioning

confidence: 99%

Frequent hypomethylation in multiple promoter CpG islands is associated with global hypomethylation, but not with frequent promoter hypermethylation

et al. 2004

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Hypomethylation of the global genome, considered to be composed mainly of repetitive sequences, is consistently observed in cancers, and aberrant hypo-and hypermethylation of CpG islands (CGIs) in promoter regions are also observed. Since methylation alterations in unique promoter sequences and in other genomic regions have distinct consequences, we analyzed the relationship between the global hypomethylation and the hypomethylation of unique promoter CGIs using human gastric cancers. Seven of ten gastric cancer cell lines showed marked decreases in 5-methylcytosine content, which correlated with hypomethylation of the LINE1 repetitive sequence. Six of the seven cell lines showed hypomethylation in five or all of the six normally methylated CGIs in promoter regions of six genes, and this was associated with induction of aberrant expression. The remaining three cell lines without global hypomethylation showed promoter hypomethylation in one or none of the six CGIs. Frequent promoter hypomethylation, however, did not correlate with frequent promoter hypermethylation. In primary gastric cancers too, global hypomethylation was associated with hypomethylation of LINE1 repetitive sequence and promoter hypomethylation. Of 93 gastric cancers, 33 cancers with frequent promoter hypomethylation and 27 cancers with frequent promoter hypermethylation constituted different groups. These findings represent experimental evidence that frequent hypomethylation of normally methylated promoter CGIs is associated with global hypomethylation, and that these hypomethylations occur independently of frequent promoter CGI hypermethylation. (Cancer Sci 2004; 95: 58-64) berrant methylation of various genomic regions is present in cancers. Firstly, global hypomethylation, the decrease of 5-methylcytosine content in the genome, 1) is known to involve coding regions of genes 2) and repetitive sequences.3, 4) Based on the facts that 80% of CpG dinucleotides are present in repetitive sequences and they are mostly methylated, 5,6) global hypomethylation is considered to be mainly due to hypomethylation of repetitive sequences.Secondly, hypomethylation is also observed in normally methylated CpG islands (CGIs) in promoter regions, and it induces aberrant expression of their downstream genes if transcription factors are available in cancer cells. Known normally methylated CGIs are very limited, and include some cancer-testis antigen genes, such as the MAGE genes.7-9) Hypomethylation of promoter CGIs is considered to have little effect on the content of 5-methylcytosine in the genome, and is distinct from hypomethylation of the repetitive sequences in the sense that it has a direct function on gene expression. While global hypomethylation is observed in most cancers, 1, 10) aberrant MAGE gene expressions were reported to occur in a smaller proportion of cancer cases (ranging from 0% to 86%, but generally from 10% to 40%).11) Therefore, a distinct regulatory mechanism may function for normally methylated promoter CGIs, and their hypomethylation m...

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Section: Methodsmentioning

confidence: 99%

Frequent hypomethylation in multiple promoter CpG islands is associated with global hypomethylation, but not with frequent promoter hypermethylation

et al. 2004

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“…High-pressure liquid-chromatography DNA samples were digested by nuclease P1 (Boehringer Mannheim, Indianapolis, IN, USA) and alkaline phosphatase (Promega, Madison, WI, USA) for 4 h at 37°C (Gehrke et al, 1984). Samples were centrifuged, and 25-to 200-p1 aliquots of the supernatants were injected directly into a HPLC apparatus (purchased from Waters, Milford, MA, USA), equipped with a 440 Absorbance Detector and a 10 cm Brownlee RP-18 column guarded by a Brownlee Aquaphore ODS precolumn.…”

Section: Materials and Methods Source And Processing Of Tissue Specimensmentioning

confidence: 99%

Alterations in DNA methylation are early, but not initial, events in ovarian tumorigenesis

Cheng

Schmütte²,

Cofer³

et al. 1997

Br J Cancer

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Summary We compared global levels of DNA methylation as well as methylation of a specific locus (MyoDl) in ovarian cystadenomas, ovarian tumours of low malignant potential (LMP) and ovarian carcinomas to investigate the association between changes in DNA methylation and ovarian tumour development. As we realized that cystadenomas showed different methylation patterns from both LMP tumours and carcinomas, we verified their monoclonal origin as a means of confirming their true neoplastic nature. High-pressure liquid chromatographic (HPLC) analyses showed that global methylation levels in LMP tumours and carcinomas were 21% and 25% lower than in cystadenomas respectively (P = 0.0001 by one-way variance analysis). Changes in the methylation status of the MyoDl locus were not seen in any of ten cystadenomas analysed but were present in five of ten LMP tumours and in five of ten carcinomas (P = 0.03). These findings suggest that alterations in DNA methylation are absent (or at least not as extensive) in ovarian cystadenomas, but are present in LMP tumours, the phenotypic features of which are intermediate between those of benign and malignant ovarian tumours. The results also emphasize the merit of distinguishing ovarian LMP tumours from cystadenomas, in spite of their similar clinical characteristics.

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“…Subsequently, 10 l of 1 M Tris and 4 units of FastAP (Fermentas) were added, and the mixture was incubated for an additional 16 h at 37°C. Nucleoside analysis was performed as described by Gehrke et al (30) using a LiChrospher 100 RP 18 column (5 m, 250 ϫ 4.6 mm).…”

Section: Mocs3-catalyzed Adenylation Of Mocs2a and Urm1mentioning

confidence: 99%

Dual Role of the Molybdenum Cofactor Biosynthesis Protein MOCS3 in tRNA Thiolation and Molybdenum Cofactor Biosynthesis in Humans

Chowdhury

Dosche

Löhmannsröben

et al. 2012

Journal of Biological Chemistry

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Background: E1-like proteins are required for activation and thiocarboxylation of ␤-grasp fold proteins involved in sulfur transfer to cofactors and tRNA. Results: MOCS3 interacts with both URM1 and MOCS2A in vivo and in vitro. Conclusion: Molybdenum cofactor biosynthesis and tRNA thiolation steps are linked by the MOCS3 protein in humans. Significance: The studies contribute to understanding the mechanism of protein conjugation and thiocarboxylate formation in sulfur transfer pathways.

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Quantitative reversed-phase high-performance liquid chromatography of major and modified nucleosides in dna

Cited by 200 publications

References 26 publications

Frequent hypomethylation in multiple promoter CpG islands is associated with global hypomethylation, but not with frequent promoter hypermethylation

Frequent hypomethylation in multiple promoter CpG islands is associated with global hypomethylation, but not with frequent promoter hypermethylation

Alterations in DNA methylation are early, but not initial, events in ovarian tumorigenesis

Dual Role of the Molybdenum Cofactor Biosynthesis Protein MOCS3 in tRNA Thiolation and Molybdenum Cofactor Biosynthesis in Humans

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