Ivarie R, Morris J A
Mol Cell Biol. 1986 Jan;6(1):97-104. doi: 10.1128/mcb.6.1.97-104.1986.
HeLA H23 cells are a mutant female human tumor cell line harboring defective hypoxanthine phosphoribosyltransferase (HPRT; IMP-pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) as a result of a mutation that alters the isoelectric point of the enzyme (G. Milman, E. Lee, G. S. Changas, J. R. McLaughlin, and J. George, Jr., Proc. Natl. Acad. Sci. USA 73:4589-4592, 1976). As shown by Milman et al. and confirmed by us here, rare HAT+ revertants arise spontaneously at 1.9 X 10(-8) frequency and express both mutant and wild-type polypeptides. Thus, the H23 mutant also carries a silent wild-type HPRT allele that is activated in revertants. To test whether the silent allele was activated via hypomethylation of genomic DNA, H23 cells were treated with inhibitors of DNA methylation, and revertants were scored by HAT or azaserine selection. At an optimal dose of 5 microM 5-azacytidine, the reversion frequency was increased about 50-fold when assayed by HAT selection and over 1,000-fold when assayed by azaserine selection. HAT+ and azaserine revertants were heterozygous for HPRT, expressing both wild-type and mutant HPRT polypeptides. Like spontaneous revertants, they contained active HPRT enzyme and were genetically unstable, reverting at about 10(-4) frequency. Similar results were found after treatment with N-methyl-N'-nitro-N-nitrosoguanidine, a DNA-alkylating agent and potent inhibitor of mammalian DNA methylation. By contrast, the DNA-ethylating agent, ethyl methanesulfonate (EMS), did not increase the HAT+ reversion frequency; it did, however, increase the frequency by which H23 revertants heterozygous for HPRT reverted to 6-thioguanine resistance. Of nine EMS revertants, seven lacked HPRT activity and had a substantially reduced expression of the wild-type polypeptide. These observations support the hypothesis that DNA methylation plays an important role in human X-chromosome inactivation and that EMS can inactivate gene expression by promoting enzymatic methylation of genomic DNA as found previously for the prolactin gene in GH3 rat pituitary tumor cells (R. D. Ivarie and J. A. Morris, Proc. Natl. Acad. Sci. USA 79:2967-2970, 1982; R. D. Ivarie, J. A. Morris, and J. A. Martial, Mol. Cell. Biol. 2:179-189, 1982).
HeLA H23细胞是一种突变的女性人类肿瘤细胞系，由于发生了改变该酶等电点的突变，其次黄嘌呤磷酸核糖转移酶（HPRT；IMP - 焦磷酸磷酸核糖转移酶，EC 2.4.2.8）存在缺陷（G. 米尔曼、E. 李、G. S. 钱加斯、J. R. 麦克劳克林和小J. 乔治，《美国国家科学院院刊》73:4589 - 4592，1976年）。如米尔曼等人所示并经我们在此证实，罕见的HAT + 回复突变体以1.9×10⁻⁸的频率自发产生，并表达突变型和野生型多肽。因此，H23突变体还携带一个沉默的野生型HPRT等位基因，该等位基因在回复突变体中被激活。为了测试沉默等位基因是否通过基因组DNA的去甲基化而被激活，用DNA甲基化抑制剂处理H23细胞，并通过HAT或氮杂丝氨酸选择对回复突变体进行评分。在5微摩尔5 - 氮杂胞苷的最佳剂量下，通过HAT选择测定时回复频率增加了约50倍，通过氮杂丝氨酸选择测定时增加了1000倍以上。HAT + 和氮杂丝氨酸回复突变体对于HPRT是杂合的，表达野生型和突变型HPRT多肽。与自发回复突变体一样，它们含有活性HPRT酶，并且遗传不稳定，回复频率约为10⁻⁴。在用N - 甲基 - N'- 硝基 - N - 亚硝基胍（一种DNA烷基化剂和哺乳动物DNA甲基化的有效抑制剂）处理后也发现了类似结果。相比之下，DNA乙基化剂甲磺酸乙酯（EMS）并没有增加HAT + 回复频率；然而，它确实增加了H23中对HPRT杂合的回复突变体对6 - 硫鸟嘌呤抗性的回复频率。在九个EMS回复突变体中，七个缺乏HPRT活性，并且野生型多肽的表达大幅降低。这些观察结果支持了这样的假设，即DNA甲基化在人类X染色体失活中起重要作用，并且EMS可以通过促进基因组DNA的酶促甲基化来使基因表达失活，正如先前在GH3大鼠垂体肿瘤细胞中的催乳素基因所发现的那样（R. D. 伊瓦里和J. A. 莫里斯，《美国国家科学院院刊》79:2967 - 2970，1982年；R. D. 伊瓦里、J. A. 莫里斯和J. A. 马蒂亚尔，《分子细胞生物学》2:179 - 189，1982年）。
