Qu Jing, Kumar Avinash, Liu Yi-Ming, Odubanjo Oluwatoyin V, Noubissi Felicite K, Hu Yixin, Hu Hankun
Jackson State University, Department of Chemistry, Physics and Atmospheric Science, Jackson, Mississippi 39217, United States.
Department of Biology, Jackson State University, Jackson, Mississippi 39217, United States.
ACS Omega. 2025 Mar 25;10(13):13370-13376. doi: 10.1021/acsomega.4c11277. eCollection 2025 Apr 8.
Measuring DNA cytosine methylation excretion presents challenges because methylated cytosine species are released in various forms including free molecules and those bound in DNA fragments. Herein, we report a novel UPLC-MS/MS method that allows the quantification of both free and DNA fragment-bound forms of methylated cytosine species excreted, providing total amounts for each. Cell culture medium and genomic DNA isolated from cells are analyzed to quantify methylated cytosine species. In genomic DNA isolated from MDA-MB-231 breast cancer cells, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are detected at 5.1% and 0.07% of total cytosine residues, respectively. In the cell culture medium, only 5hmC is detected at a low level (ca. 7 nM). However, in two normal cell lines (i.e., primary mouse lung epithelial cells and HEK293 kidney cells) 5mC, 5-methylcytidine, and 2'-oxymethylcytidine (but no 5hmC) are found present in cell culture medium at concentrations ranging from 10 to 320 nM. Further, it is observed for the first time that treating MDA-MB-231 cells with carboplatin significantly increases the 5hmC level in the culture medium, indicating a carboplatin-boosted DNA cytosine methylation excretion from cancer cells.
测量DNA胞嘧啶甲基化排泄存在挑战，因为甲基化胞嘧啶物种以多种形式释放，包括游离分子和与DNA片段结合的分子。在此，我们报告了一种新颖的超高效液相色谱-串联质谱法，该方法能够对排泄的甲基化胞嘧啶物种的游离形式和与DNA片段结合的形式进行定量，从而提供每种形式的总量。对细胞培养基和从细胞中分离的基因组DNA进行分析，以定量甲基化胞嘧啶物种。在从MDA-MB-231乳腺癌细胞中分离的基因组DNA中，5-甲基胞嘧啶（5mC）和5-羟甲基胞嘧啶（5hmC）分别占总胞嘧啶残基的5.1%和0.07%。在细胞培养基中，仅检测到低水平（约7 nM）的5hmC。然而，在两种正常细胞系（即原代小鼠肺上皮细胞和HEK293肾细胞）的细胞培养基中发现存在浓度范围为10至320 nM的5mC、5-甲基胞苷和2'-氧甲基胞苷（但未检测到5hmC）。此外，首次观察到用卡铂处理MDA-MB-231细胞会显著提高培养基中的5hmC水平，表明卡铂促进了癌细胞的DNA胞嘧啶甲基化排泄。
