DNA (Cytosine-C5) methyltransferase inhibition by oligodeoxyribonucleotides containing 2-(1H)-pyrimidinone (zebularine aglycon) at the enzymatic target site

20 pages, 7 figures, 1 table.-- PMID: 19467223 [PubMed].-- PMCID: PMC2756644.-- NIHMSID: NIHMS130041.-- Printed version published Sep 15, 2009.Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer progression. A number of DNA methyltransferase inhibitors are known to reactivate silenced genes; including 5-azacytidine and 2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic basis for this difference, we carried out a detailed comparisons of the interaction between purified DNA methyltransferases and oligodeoxyribonucleotides (ODNs) containing either 5-azacytosine or 2-(1H)-pyrimidinone in place of the cytosine targeted for methylation. When incorporated into small ODNs, the rate of C5 DNA methyltransferase inhibition by both nucleosides is essentially identical. However, the stability and reversibility of the enzyme complex in the absence and presence of cofactor differs. 5-Azacytosine ODNs form complexes with C5 DNA methyltransferases that are irreversible when the 5-azacytosine ring is intact. ODNs containing 2-(1H)-pyrimidinone at the enzymatic target site are competitive inhibitors of both prokaryotic and mammalian DNA C5 methyltransferases. We determined that the ternary complexes between the enzymes, 2-(1H)-pyrimidinone inhibitor, and the cofactor S-adenosyl methionine are maintained through the formation of a reversible covalent interaction. The differing stability and reversibility of the covalent bonds may partially account for the observed differences in cytotoxicity between zebularine and 5-azacytidine inhibitors.Partial support for this work was provided by a grant from the NIH/NCI (R21CA91315) to J.K.C. and a fellowship from the Graduate College at UNMC to D.V.B. We are grateful to S. Kumar of New England Biolabs for providing us with Eschericia coli strain ER1727 containing the pUHE25HhaI plasmid. This research was also supported in part with funds from the Intramural Research Program of the NIH, Center for Cancer Research, NCI Frederick.Peer reviewe

DNA (Cytosine-C5) methyltransferase inhibition by oligodeoxyribonucleotides containing 2-(1H)-pyrimidinone (zebularine aglycon) at the enzymatic target site

20 pages, 7 figures, 1 table.-- PMID: 19467223 [PubMed].-- PMCID: PMC2756644.-- NIHMSID: NIHMS130041.-- Printed version published Sep 15, 2009.Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer progression. A number of DNA methyltransferase inhibitors are known to reactivate silenced genes; including 5-azacytidine and 2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic basis for this difference, we carried out a detailed comparisons of the interaction between purified DNA methyltransferases and oligodeoxyribonucleotides (ODNs) containing either 5-azacytosine or 2-(1H)-pyrimidinone in place of the cytosine targeted for methylation. When incorporated into small ODNs, the rate of C5 DNA methyltransferase inhibition by both nucleosides is essentially identical. However, the stability and reversibility of the enzyme complex in the absence and presence of cofactor differs. 5-Azacytosine ODNs form complexes with C5 DNA methyltransferases that are irreversible when the 5-azacytosine ring is intact. ODNs containing 2-(1H)-pyrimidinone at the enzymatic target site are competitive inhibitors of both prokaryotic and mammalian DNA C5 methyltransferases. We determined that the ternary complexes between the enzymes, 2-(1H)-pyrimidinone inhibitor, and the cofactor S-adenosyl methionine are maintained through the formation of a reversible covalent interaction. The differing stability and reversibility of the covalent bonds may partially account for the observed differences in cytotoxicity between zebularine and 5-azacytidine inhibitors.Partial support for this work was provided by a grant from the NIH/NCI (R21CA91315) to J.K.C. and a fellowship from the Graduate College at UNMC to D.V.B. We are grateful to S. Kumar of New England Biolabs for providing us with Eschericia coli strain ER1727 containing the pUHE25HhaI plasmid. This research was also supported in part with funds from the Intramural Research Program of the NIH, Center for Cancer Research, NCI Frederick.Peer reviewe

LINE-1 Methylation Levels in Leukocyte DNA and Risk of Renal Cell Cancer

Leukocyte global DNA methylation levels are currently being considered as biomarkers of cancer susceptibility and have been associated with risk of several cancers. In this study, we aimed to examine the association between long interspersed nuclear elements (LINE-1) methylation levels, as a biomarker of global DNA methylation in blood cell DNA, and renal cell cancer risk.LINE-1 methylation of bisulfite-converted genomic DNA isolated from leukocytes was quantified by pyrosequencing measured in triplicate, and averaged across 4 CpG sites. A total of 328 RCC cases and 654 controls frequency-matched(2∶1) on age(±5years), sex and study center, from a large case-control study conducted in Central and Eastern Europe were evaluated.LINE-1 methylation levels were significantly higher in RCC cases with a median of 81.97% (interquartile range[IQR]: 80.84–83.47) compared to 81.67% (IQR: 80.35–83.03) among controls (p = 0.003, Wilcoxon). Compared to the lowest LINE-1 methylation quartile(Q1), the adjusted ORs for increasing methylation quartiles were as follows: OR(Q2) = 1.84(1.20−2.81), OR(Q3) = 1.72(1.11−2.65) and OR(Q4) = 2.06(1.34−3.17), with a p-trend = 0.004. The association was stronger among current smokers (p-trend<0.001) than former or never smokers (p-interaction = 0.03). To eliminate the possibility of selection bias among controls, the relationship between LINE-1 methylation and smoking was evaluated and confirmed in a case-only analysis, as well.Higher levels of LINE-1 methylation appear to be positively associated with RCC risk, particularly among current smokers. Further investigations using both post- and pre-diagnostic genomic DNA is warranted to confirm findings and will be necessary to determine whether the observed differences occur prior to, or as a result of carcinogenesis

Comprehensive Analysis of 5-Aminolevulinic Acid Dehydrogenase (ALAD) Variants and Renal Cell Carcinoma Risk among Individuals Exposed to Lead

BACKGROUND: Epidemiologic studies are reporting associations between lead exposure and human cancers. A polymorphism in the 5-aminolevulinic acid dehydratase (ALAD) gene affects lead toxicokinetics and may modify the adverse effects of lead. METHODS: The objective of this study was to evaluate single-nucleotide polymorphisms (SNPs) tagging the ALAD region among renal cancer cases and controls to determine whether genetic variation alters the relationship between lead and renal cancer. Occupational exposure to lead and risk of cancer was examined in a case-control study of renal cell carcinoma (RCC). Comprehensive analysis of variation across the ALAD gene was assessed using a tagging SNP approach among 987 cases and 1298 controls. Occupational lead exposure was estimated using questionnaire-based exposure assessment and expert review. Odds ratios (OR) and 95% confidence intervals (CI) were calculated using logistic regression. RESULTS: The adjusted risk associated with the ALAD variant rs8177796(CT/TT) was increased (OR = 1.35, 95%CI = 1.05-1.73, p-value = 0.02) when compared to the major allele, regardless of lead exposure. Joint effects of lead and ALAD rs2761016 suggest an increased RCC risk for the homozygous wild-type and heterozygous alleles ((GG)OR = 2.68, 95%CI = 1.17-6.12, p = 0.01; (GA)OR = 1.79, 95%CI = 1.06-3.04 with an interaction approaching significance (p(int) = 0.06). No significant modification in RCC risk was observed for the functional variant rs1800435(K68N). Haplotype analysis identified a region associated with risk supporting tagging SNP results. CONCLUSION: A common genetic variation in ALAD may alter the risk of RCC overall, and among individuals occupationally exposed to lead. Further work in larger exposed populations is warranted to determine if ALAD modifies RCC risk associated with lead exposure

Comparing algorithms for automated vessel segmentation in computed tomography scans of the lung: the VESSEL12 study

The VESSEL12 (VESsel SEgmentation in the Lung) challenge objectively compares the performance of different algorithms to identify vessels in thoracic computed tomography (CT) scans. Vessel segmentation is fundamental in computer aided processing of data generated by 3D imaging modalities. As manual vessel segmentation is prohibitively time consuming, any real world application requires some form of automation. Several approaches exist for automated vessel segmentation, but judging their relative merits is difficult due to a lack of standardized evaluation. We present an annotated reference dataset containing 20 CT scans and propose nine categories to perform a comprehensive evaluation of vessel segmentation algorithms from both academia and industry. Twenty algorithms participated in the VESSEL12 challenge, held at International Symposium on Biomedical Imaging (ISBI) 2012. All results have been published at the VESSEL12 website http://vessel12.grand-challenge.org. The challenge remains ongoing and open to new participants. Our three contributions are: (1) an annotated reference dataset available online for evaluation of new algorithms; (2) a quantitative scoring system for objective comparison of algorithms; and (3) performance analysis of the strengths and weaknesses of the various vessel segmentation methods in the presence of various lung diseases.Rudyanto, RD.; Kerkstra, S.; Van Rikxoort, EM.; Fetita, C.; Brillet, P.; Lefevre, C.; Xue, W.... (2014). Comparing algorithms for automated vessel segmentation in computed tomography scans of the lung: the VESSEL12 study. Medical Image Analysis. 18(7):1217-1232. doi:10.1016/j.media.2014.07.003S1217123218