TOXICITY OF 5-AZA-2'-DEOXYCYTIDINE TO MAMMALIAN-CELLS IS MEDIATED PRIMARILY BY COVALENT TRAPPING OF DNA METHYLTRANSFERASE RATHER THAN DNA DEMETHYLATION
TOXICITY OF 5-AZA-2'-DEOXYCYTIDINE TO MAMMALIAN-CELLS IS MEDIATED PRIMARILY BY COVALENT TRAPPING OF DNA METHYLTRANSFERASE RATHER THAN DNA DEMETHYLATION
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DOI：
10.1073/pnas.91.25.11797
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发表时间：
1994-12-06
影响因子：
11.1
通讯作者：
JAENISCH, R
中科院分区：
文献类型：
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作者：
JUTTERMANN, R;LI, E;JAENISCH, R
脱氧胞苷类似物5-氮杂-2‘-脱氧胞苷(5-azadCyd)被广泛用作DNA甲基化抑制剂，通过实验诱导基因表达和细胞分化。在出现DNA甲基转移酶水平改变的突变小鼠之前，用药物处理细胞一直是在实验上操纵哺乳动物细胞基因组DNA甲基化水平的唯一手段。用5-azadCyd取代DNA会导致酶的共价捕获，从而耗尽细胞的酶活性，导致DNA去甲基化。5-azadCyd或5-azacytidine处理会引起细胞的多种变化，包括沉默基因的激活，染色质的解缩，以及细胞分化的诱导，所有这些都被认为是药物诱导去甲基化的结果。5-azadCyd在培养的细胞和动物中具有高度毒性，被用作治疗某些人类癌症的有效抗肿瘤药物。据推测，该药物对哺乳动物细胞的毒性也是由于它抑制了DNA甲基化。然而，甲基化反应的化学成分是一致的，但有另一种机制：5-azadCyd的细胞毒作用可能是通过DNA甲基转移酶与5-azadCyd取代的DNA的共价结合直接介导的。我们通过使用胚胎干细胞和由于该基因的靶向突变而导致DNA甲基转移酶水平降低的小鼠来测试这种可能性。当暴露于5-azadCyd突变的胚胎干细胞或胚胎时，对药物的毒性作用的抵抗力分别显著高于野生型细胞和胚胎。这些结果有力地表明，细胞DNA甲基转移酶本身，而不是基因组DNA的次级去甲基化，是5-azadCyd细胞毒性的主要中介。根据我们的结果，以前使用5-azadCyd进行实验操作细胞甲基化水平的研究得出的一些结论可能需要重新评估。此外，我们的数据为癌症治疗提供了明确的预测：DNA甲基转移酶水平升高的肿瘤细胞预计将对5-azadCyd治疗敏感，而酶水平降低的肿瘤将具有耐药性。
The deoxycytidine analog 5-aza-2'-deoxycytidine (5-azadCyd) has been widely used as a DNA methylation inhibitor to experimentally induce gene expression and cellular differentiation. Prior to the availability of mutant mice with altered DNA methyltransferase levels, treatment of cells with drugs has been the only means to experimentally manipulate the level of genomic DNA methylation in mammalian cells. Substitution of DNA with 5-azadCyd leads to covalent trapping of the enzyme, thereby depleting the cells of enzyme activity and resulting in DNA demethylation. 5-AzadCyd or 5-azacytidine treatment causes multiple changes in treated cells, including activation of silent genes, decondensation of chromatin, and induction of cellular differentiation, all of which are believed to be consequences of drug-induced demethylation. 5-AzadCyd is highly toxic in cultured cells and animals and is utilized as a potent antitumor agent for treatment of certain human cancers. It has been postulated that the toxicity of the drug in mammalian cells is also due to its inhibition of DNA methylation. The chemistry of the methylation reaction is consistent, however, with an alternative mechanism: the cytotoxic effect of 5-azadCyd may be directly mediated through the covalent binding of DNA methyltransferase to 5-azadCyd-substituted DNA. We have tested this possibility by using embryonic stem cells and mice with reduced levels of DNA methyltransferase due to a targeted mutation of the gene. When exposed to 5-azadCyd mutant embryonic stern cells or embryos were significantly more resistant to the toxic effects of the drug than wild-type cells and embryos, respectively. These results strongly suggest that the cellular DNA methyltransferase itself, rather than the secondary demethylation of genomic DNA, is the primary mediator of 5-azadCyd cytotoxicity. In light of our results, some conclusions from previous studies using 5-azadCyd in order to experimentally manipulate cellular methylation levels may have to be reassessed. Also, our data make clear predictions for cancer treatment: tumor cells with elevated DNA methyltransferase levels would be expected to be susceptible to treatment with 5-azadCyd, whereas tumors with reduced levels of the enzyme would be resistant.