Boffa L C, Mariani M R, Parker M I
Department of Chemical Carcinogenesis, Istituto Nazionale per la Ricerca sul Cancro, IST, Genova, Italy.
Exp Cell Res. 1994 Apr;211(2):420-3. doi: 10.1006/excr.1994.1107.
Previous studies have shown that treatment of cultured fibroblasts with millimolar concentrations of sodium butyrate results in increased methylation of cytosine residues in DNA. In this study, active nucleosomes were fractionated from the inactive ones by organomercurial agarose column chromatography. DNA in each fraction was hydrolyzed to its constituent bases and subjected to HPLC analysis in order to determine the 5-methylcytosine content. In control cells, the active nucleosomal DNA was hypomethylated (0.97 +/- 0.27% 5-methyleytosine) when compared with the inactive DNA fraction (1.61 +/- 0.15%). This result was not unexpected since DNA hypermethylation is generally associated with gene inactivation. Treatment of cells with sodium butyrate, however, resulted in increased methylation of the active nucleosomal DNA such that it was comparable to that of the inactive fraction of control cells (1.73 +/- 0.02% 5-methylcytosine). A much smaller increase in 5-methylcytosine content was detected in the inactive DNA fraction of sodium butyrate-treated cells (from 1.61 to 1.89%). Removal of the sodium butyrate followed by a chase in butyrate-free medium for up to 120 h failed to reverse the butyrate-induced hypermethylation. Reversal was achieved only after continuous culture in butyrate-free medium for 10 days.
先前的研究表明，用毫摩尔浓度的丁酸钠处理培养的成纤维细胞会导致DNA中胞嘧啶残基的甲基化增加。在本研究中，通过有机汞琼脂糖柱色谱法将活性核小体与非活性核小体分离。将每个组分中的DNA水解成其组成碱基，并进行HPLC分析以确定5-甲基胞嘧啶含量。在对照细胞中，与非活性DNA组分（1.61±0.15%）相比，活性核小体DNA的甲基化程度较低（0.97±0.27% 5-甲基胞嘧啶）。这一结果并不意外，因为DNA高甲基化通常与基因失活有关。然而，用丁酸钠处理细胞会导致活性核小体DNA的甲基化增加，使其与对照细胞的非活性组分相当（1.73±0.02% 5-甲基胞嘧啶）。在丁酸钠处理的细胞的非活性DNA组分中检测到5-甲基胞嘧啶含量的增加要小得多（从1.61%增加到1.89%）。去除丁酸钠后，在无丁酸盐的培养基中追踪长达120小时未能逆转丁酸盐诱导的高甲基化。只有在无丁酸盐的培养基中连续培养10天后才能实现逆转。
