Cytological evidence for 5-azacytidine-induced demethylation of the heterochromatic regions of human chromosomes
Cytological evidence for 5-azacytidine-induced demethylation of the heterochromatic regions of human chromosomes
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DOI：
10.1007/bf02263676
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发表时间：
1996-06-01
影响因子：
2.6
通讯作者：
Niveleau, A
中科院分区：
文献类型：
--
作者：
deCapoa, A;Menendez, F;Niveleau, A
本工作旨在从细胞学水平研究去甲基化试剂5-氮杂胞苷(5-azaC)对人染色体结构异染色质的影响。用标准方法获得的外周血淋巴细胞和淋巴母细胞培养的中期制剂用该试剂处理。异染色区的标记采用抗5-甲基胞嘧啶(5MeC)单抗和过氧化物酶标记的第二抗体的间接免疫染色方法。底物为4-氯-1-萘酚(4C1N)。用抗5MeC抗体与特定染色体区域结合的频率来衡量单个染色体或染色体组的甲基化率。获得的结果如下：(I)在对照培养中，抗5MeC抗体与顶端着丝粒短臂区域的结合频率在个体内和个体间都有很高的变异性；(Ii)5-azaC始终影响构成异染色质的甲基化状态；(Iii)在特定染色体的异染色质区域发生优先去甲基化；(Iv)在我们的实验条件下，5-azaC的去甲基化作用似乎与该药物众所周知的解卷效应无关。
This work was aimed at studying the effects of the demethylating agent 5-azacytidine (5-azaC) on the constitutive heterochromatin of human chromosomes at the cytological level. Metaphase preparations from peripheral blood lymphocyte and lymphoblastoid cultures obtained by standard methods were treated with the agent. Labelling of the heterochromatic regions was achieved by the indirect immunostaining method using anti-5-methylcytosine (5MeC) monoclonal antibodies and peroxidase-tagged second antibodies. 4-Chloro-1-naphthol (4C1N) was used as the substrate. The rate of methylation of individual chromosomes or chromosome groups was measured as the frequency of binding of anti-5MeC antibodies to specific chromosome regions. The following results were obtained: (i) in control cultures high intra- and interindividual variability in the binding frequencies of anti-5MeC antibodies to the short arm region of the acrocentrics was observed; (ii) 5-azaC consistently affects the methylation status of the constitutive heterochromatin; (iii) preferential demethylation occurs in the heterochromatic regions of specific chromosomes; (iv) under our experimental conditions the demethylating effect of 5-azaC appears not to be related to the well-known uncoiling effect of this drug.