Dataset Information

Accurate quantification of 5-Methylcytosine, 5-Hydroxymethylcytosine, 5-Formylcytosine, and 5-Carboxylcytosine in genomic DNA from breast cancer by chemical derivatization coupled with ultra performance liquid chromatography- electrospray quadrupole time of flight mass spectrometry analysis.

ABSTRACT: The DNA demethylation pathway has been discovered to play a significant role in DNA epigenetics. This pathway removes the methyl group from cytosine, which is involved in the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) by ten-eleven translocation (TET) proteins. Then, 5-hmC can be iteratively oxidized to generate 5-formylcytosine (5-foC) and 5-carboxylcytosine (5-caC). However, 5-hmC, 5-foC, and 5-caC are hardly detected due to their low content. In this study, we have developed a LC-HRMS method coupled with derivatization to accurately and simultaneously quantify 5-mC levels, along with its oxidation products in genomic DNA. Derivatization was carried out using 4-dimethylamino benzoic anhydride, which has been shown to improve separation and enhance the detection sensitivity. Finally, we successfully applied this method towards the quantification of 5-mC, 5-hmC, 5-foC, and 5-caC in genomic DNA isolated from both human breast cancer tissue and tumor-adjacent normal tissue. We show that 5-foC and 5-caC are increased in tumor tissue. In addition, the levels of 5-mC, 5-hmC, 5-foC, and 5-caC measured in tumor tissue versus tumor-adjacent tissue were found to be distinct among different classifications. This suggests that cytosine modifiers could be used as potential biomarkers for determining the stage of development of breast cancer, as well as prognosis.

SUBMITTER: Guo M

PROVIDER: S-EPMC5710920 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundPrognostic tools for prostate cancer (PC) are inadequate and new molecular biomarkers may improve risk stratification. The epigenetic mark 5-hydroxymethylcytosine (5hmC) has recently been proposed as a novel candidate prognostic biomarker in several malignancies including PC. 5hmC is an oxidized derivative of 5-methylcytosine (5mC) and can be further oxidized to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The present study is the first to investigate the biomarker potential in PC for all four DNA methylation marks in parallel. Thus, we determined 5mC, 5hmC, 5fC, and 5caC levels in non-malignant (NM) and PC tissue samples from a large radical prostatectomy (RP) patient cohort (n = 546) by immunohistochemical (IHC) analysis of serial sections of a tissue microarray. Possible associations between methylation marks, routine clinicopathological parameters, ERG status, and biochemical recurrence (BCR) after RP were investigated.Results5mC and 5hmC levels were significantly reduced in PC compared to NM prostate tissue samples (p ≤ 0.027) due to a global loss of both marks specifically in ERG- PCs. 5fC levels were significantly increased in ERG+ PCs (p = 0.004), whereas 5caC levels were elevated in both ERG- and ERG+ PCs compared with NM prostate tissue samples (p ≤ 0.019). Positive correlations were observed between 5mC, 5fC, and 5caC levels in both NM and PC tissues (p < 0.001), while 5hmC levels were only weakly positively correlated to 5mC in the PC subset (p = 0.030). There were no significant associations between 5mC, 5fC, or ERG status and time to BCR in this RP cohort. In contrast, high 5hmC levels were associated with BCR in ERG- PCs (p = 0.043), while high 5caC levels were associated with favorable prognosis in ERG+ PCs (p = 0.011) and were borderline significantly associated with worse prognosis in ERG- PCs (p = 0.058). Moreover, a combined high-5hmC/high-5caC score was a significant adverse predictor of post-operative BCR beyond routine clinicopathological variables in ERG- PCs (hazard ratio 3.18 (1.54-6.56), p = 0.002, multivariate Cox regression).ConclusionsThis is the first comprehensive study of 5mC, 5hmC, 5fC, and 5caC levels in PC and the first report of a significant prognostic potential for 5caC in PC.

Project description:Replication-independent active/enzymatic demethylation may be an important process in the functioning of somatic cells. The most plausible mechanisms of active 5-methylcytosine demethylation, leading to activation of previously silenced genes, involve ten-eleven translocation (TET) proteins that participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxylcytosine. Recently, 5-hydroxymethylcytosine was demonstrated to be a relatively stable modification, and the previously observed substantial differences in the level of this modification in various murine tissues were shown to depend mostly on cell proliferation rate. Some experimental evidence supports the hypothesis that 5-hydroxymethyluracil may be also generated by TET enzymes and has epigenetic functions.Using an isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry, we have analyzed, for the first time, all the products of active DNA demethylation pathway: 5-methyl-2'-deoxycytidine, 5-hydroxymethyl-2'-deoxycytidine, 5-formyl-2'-deoxycytidine and 5-carboxyl-2'-deoxycytidine, as well as 5-hydroxymethyl-2'-deoxyuridine, in DNA isolated from various rat and porcine tissues. A strong significant inverse linear correlation was found between the proliferation rate of cells and the global level of 5-hydroxymethyl-2'-deoxycytidine in both porcine (R2 = 0.88) and rat tissues (R2 = 0.83); no such relationship was observed for 5-formyl-2'-deoxycytidine and 5-carboxyl-2'-deoxycytidine. Moreover, a substrate-product correlation was demonstrated for the two consecutive steps of iterative oxidation pathway: between 5-hydroxymethyl-2'-deoxycytidine and its product 5-formyl-2'-deoxycytidine, as well as between 5-formyl-2'-deoxycytidine and 5-carboxyl-2'-deoxycytidine (R2 = 0.60 and R2 = 0.71, respectively).Good correlations within the substrate-product sets of iterative oxidation pathway may suggest that a part of 5-formyl-2'-deoxycytidine and/or 5-carboxyl-2'-deoxycytidine can be directly linked to a small portion of 5-hydroxymethyl-2'-deoxycytidine which defines the active demethylation process.

Dataset Information

Accurate quantification of 5-Methylcytosine, 5-Hydroxymethylcytosine, 5-Formylcytosine, and 5-Carboxylcytosine in genomic DNA from breast cancer by chemical derivatization coupled with ultra performance liquid chromatography- electrospray quadrupole time of flight mass spectrometry analysis.

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