Call K M, Thilly W G
Division of Toxicology, Whitaker College of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge 02139.
Mutat Res. 1991 May;248(1):101-14. doi: 10.1016/0027-5107(91)90092-3.
This paper examines the mechanism by which 5-bromodeoxyuridine (BrdUrd) induces a high frequency of transient trifluorothymidine (F3TdR)-resistant variants in the TK6 human lymphoblast cell line (a TK +/- heterozygote). This phenomenon has previously been termed 'pseudomutation' (Liber et al., 1985). We now report that 5-azacytidine (5-AzaC), an inhibitor of DNA methylation, reverses BrdUrd-induced pseudomutation in a dose-dependent manner. The inhibition by 5-AzaC is highly specific and does not appear to involve nucleotide pool perturbations. 5-AzaC inhibits the pseudomutagenic effect (transient trifluorothymidine resistance in a thymidine kinase heterozygote), but not the stable mutagenic effect (stable 6-thioguanine resistance or trifluorothymidine resistance in a hypoxanthine-guanine phosphoribosyltransferase-proficient cell) induced by BrdUrd. 5-AzaC did not affect the induction nor expression of mutation induced by several other chemical mutagens at either the tk or hgprt loci. Inhibition of pseudomutation by 5-AzaC did not appear to be caused by a number of potential confounding factors. Although significant changes in the levels of DNA methylation were detected by HPLC analysis in BrdUrd-treated cells, the dose response for inhibition of pseudomutation by 5-AzaC was correlated with a significant decrease in 5-methylcytidine levels. These results and additional data in the literature have led us to postulate a novel mechanism in which the substitution of BrdUrd in a TpG dinucleotide(s) may serve as a substrate for non-heritable methylation and hence transiently inactivate tk gene expression.
本文研究了5-溴脱氧尿苷（BrdUrd）在TK6人淋巴母细胞系（一种TK+/-杂合子）中诱导高频短暂三氟胸苷（F3TdR）抗性变体的机制。这种现象先前被称为“假突变”（Liber等人，1985年）。我们现在报告，DNA甲基化抑制剂5-氮杂胞苷（5-AzaC）以剂量依赖的方式逆转BrdUrd诱导的假突变。5-AzaC的抑制作用具有高度特异性，似乎不涉及核苷酸池的扰动。5-AzaC抑制假诱变效应（胸苷激酶杂合子中的短暂三氟胸苷抗性），但不抑制BrdUrd诱导的稳定诱变效应（次黄嘌呤-鸟嘌呤磷酸核糖转移酶功能正常的细胞中的稳定6-硫鸟嘌呤抗性或三氟胸苷抗性）。5-AzaC对tk或hgprt位点上由其他几种化学诱变剂诱导的突变的诱导或表达没有影响。5-AzaC对假突变的抑制似乎不是由一些潜在的混杂因素引起的。尽管通过HPLC分析在BrdUrd处理的细胞中检测到DNA甲基化水平有显著变化，但5-AzaC抑制假突变的剂量反应与5-甲基胞苷水平的显著降低相关。这些结果以及文献中的其他数据使我们推测出一种新机制，即BrdUrd在TpG二核苷酸中的取代可能作为非遗传性甲基化的底物，从而暂时使tk基因表达失活。
