Synthesis and Characterization of Isotopically Enriched Pyrimidine Deoxynucleoside Oxidation Damage Products

LaFrancois, Christopher J.; Fujimoto, June; Sowers, Lawrence C.

doi:10.1021/tx970186o

Cited by 50 publications

(59 citation statements)

References 56 publications

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“…R indicates substituent at the 5-position of residue X that include H, F, Cl, Br, I, CH 3 , OH, CH 2 OH, and 1-propynyl to make cytosine, 5-fluorocytosine, 5-chlorocytosine, 5-bromocytosine, 5-iodocytosine, 5-methylcytosine, 5-hydroxycytosine, 5-hydroxymethylcytosine, and pdC, respectively. The 5-chlorocytosine, 5-hydroxycytosine, and 5-hydroxymethylcytosine phosphoramidites were prepared according to the methods developed by this laboratory (18,19). All other phosphoramidites used were obtained from Glen Research.…”

Section: Methodsmentioning

confidence: 99%

Endogenous Cytosine Damage Products Alter the Site Selectivity of Human DNA Maintenance Methyltransferase DNMT1

2007

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Alterations in cytosine methylation patterns are usually observed in human tumors. The consequences of altered cytosine methylation patterns include both inappropriate activation of transforming genes and silencing of tumor suppressor genes. Despite the biological effect of methylation changes, little is known about how such changes are caused. The heritability of cytosine methylation patterns from parent to progeny cells is attributed to the fidelity of the methylation-sensitive human maintenance methyltransferase DNMT1, which methylates with high specificity the unmethylated strand of a hemimethylated CpG sequence following DNA replication. We have been studying DNA damage that might alter the specificity of DNMT1, either inhibiting the methylation of hemimethylated sites or triggering the inappropriate methylation of previously unmethylated sites. Here, we show that known forms of endogenous DNA damage can cause either hypermethylation or hypomethylation. Inflammation-induced 5-halogenated cytosine damage products, including 5-chlorocytosine, mimic 5-methylcytosine and induce inappropriate DNMT1 methylation within a CpG sequence. In contrast, oxidation damage of the methyl group of 5-methylcytosine, with the formation of 5-hydroxymethylcytosine, prevents DNMT1 methylation of the target cytosine. We propose that reduced DNMT1 selectivity resulting from DNA damage could cause heritable changes in cytosine methylation patterns, resulting in human tumor formation. These data may provide a mechanistic link for the associations documented between inflammation and cancer. [Cancer Res 2007;67(3):946-50]

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Section: Methodsmentioning

confidence: 99%

Endogenous Cytosine Damage Products Alter the Site Selectivity of Human DNA Maintenance Methyltransferase DNMT1

2007

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“…FBS (fetal bovine serum) and dialyzed FBS were purchased from Gibco (Carlsbad, CA). (35). Synthesis of 5-methoxymethyluracil (MmU) on a milligram scale was performed according to Cline et al (36).…”

Section: Experimental Procedures Materialsmentioning

confidence: 99%

Measurement of the Incorporation and Repair of Exogenous 5-Hydroxymethyl-2′-deoxyuridine in Human Cells in Culture Using Gas Chromatography-Negative Chemical Ionization-Mass Spectrometry

Rogstad

Darwanto

Herring

et al. 2007

Chem. Res. Toxicol.

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The DNA of all organisms is constantly damaged by oxidation. Among the array of damage products is 5-hydroxymethyluracil, derived from oxidation of the thymine methyl group. Previous studies have established that HmU can be a sensitive and valuable marker of DNA damage. More recently, the corresponding deoxynucleoside, 5-hydroxymethyl-2′-deoxyuridine (HmdU) has proven to be valuable for the introduction of controlled amounts of a single type of damage lesion into the DNA of replicating cells, which is subsequently repaired by the base excision repair pathway. Complicating the study of HmU formation and repair, however, is the known chemical reactivity of the hydroxymethyl group of HmU under conditions used to hydrolyze DNA. In the work reported here, this chemical property has been exploited by creating conditions that convert HmU to the corresponding methoxymethyluracil (MmU) derivative that can be further derivatized to the 3,5-bis-(trifluoromethyl)benzyl analog. This derivatized compound can be detected by gas chromatographynegative chemical ionization-mass spectrometry (GC-NCI-MS) with good sensitivity. Using isotopically enriched exogenous HmdU and human osteosarcoma cells (U2OS) in culture, we demonstrate that this method allows for the measurement of HmU in DNA formed from incorporation of exogenous HmdU. We further demonstrate that addition of isotopically enriched uridine to the culture medium allows for the simultaneous measurement of DNA replication and repair kinetics. This sensitive and facile method should prove valuable for studies on DNA oxidation damage and repair in living cells.

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“…All other chemicals were obtained from Aldrich or VWR International (West Chester, PA). Modified nucleosides 5-formyl-2=-deoxyuridine (5-FmdU), 5-hydroxy-2=-deoxyuridine (5-OHdU), 5-formyl-2=-deoxycytidine (5-FmdC), 5-hydroxy-2=-deoxycytidine (5-OHdC), 5-hydroxymethyl-2=-deoxycytidine (5-HmdC), and all the isotopicallylabeled nucleosides (Scheme 1) were synthesized according to published procedures [20,21]. The purity and identity of the synthesized compounds were verified by HPLC, NMR, and mass spectrometry.…”

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confidence: 99%

Collisionally activated dissociation of protonated 2′-deoxycytidine, 2′-deoxyuridine, and their oxidatively damaged derivatives

Cao

Wang

2006

J. Am. Soc. Mass Spectrom.

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We examined the collisionally activated dissociation (CAD) pathways of protonated 2=-deoxycytidine (dC), 5-formyl-2=-deoxycytidine (5-FmdC), 5-hydroxy-2=-deoxycytidine (5-OHdC), 5-hydroxymethyl-2=-deoxycytidine (5-HmdC), and their corresponding stable isotope-labeled compounds to gain insights into the effects of modifications on the fragmentation pathways of the pyrimidine bases. Multi-stage MS (MS n ) results showed that protonated cytosine, its 5-hydroxyland 5-hydroxymethyl-substituted derivatives, but not its 5-formyl-substituted analog, could undergo Dimroth-like rearrangement in the gas-phase. The elimination of HNCO was one of the major fragmentation pathways observed for the protonated ions of all dC derivatives except for 5-hydroxymethylcytosine, which underwent this loss only after a H 2 O molecule had been eliminated. In addition, the protonated cytosine and 5-hydroxycytosine can undergo a facile elimination of NH 3 molecule. This loss, however, was not observed for protonated 5-hydroxymethylcytosine, 5-formylcytosine, and their uracil analogs. Taken together, our study demonstrated that modifications could alter markedly the CAD patterns of the protonated pyrimidine bases. The results from this study provided a basis for the identifications of other modified pyrimidine bases/nucleosides by tandem mass spectrometry. (J Am Soc Mass Spectrom 2006, 17, 1335-1341

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Synthesis and Characterization of Isotopically Enriched Pyrimidine Deoxynucleoside Oxidation Damage Products

Cited by 50 publications

References 56 publications

Endogenous Cytosine Damage Products Alter the Site Selectivity of Human DNA Maintenance Methyltransferase DNMT1

Endogenous Cytosine Damage Products Alter the Site Selectivity of Human DNA Maintenance Methyltransferase DNMT1

Measurement of the Incorporation and Repair of Exogenous 5-Hydroxymethyl-2′-deoxyuridine in Human Cells in Culture Using Gas Chromatography-Negative Chemical Ionization-Mass Spectrometry

Collisionally activated dissociation of protonated 2′-deoxycytidine, 2′-deoxyuridine, and their oxidatively damaged derivatives

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