Activation of a nonexpressed hypoxanthine phosphoribosyltransferase allele in mutant H23 HeLa cells by agents that inhibit DNA methylation.
Activation of a nonexpressed hypoxanthine phosphoribosyltransferase allele in mutant H23 HeLa cells by agents that inhibit DNA methylation.
复制标题
通过抑制 DNA 甲基化的药物激活突变型 H23 HeLa 细胞中非表达的次黄嘌呤磷酸核糖转移酶等位基因。
DOI：
10.1128/mcb.6.1.97-104.1986
复制
发表时间：
1986
影响因子：
5.3
通讯作者：
Morris,JA
中科院分区：
文献类型：
--
作者：
Ivarie,R;Morris,JA
HeLA H23细胞是一种突变的雌性人类肿瘤细胞系，其由于改变酶的等电点的突变而具有缺陷型次黄嘌呤磷酸核糖基转移酶（HPRT; IMP-焦磷酸磷酸核糖基转移酶，EC 2.4.2.8）（G. Milman，E.李，G. S.昌加斯McLaughlin和J.乔治，Jr.，Proc. Natl. Acad. Sci. USA 73：4589-4592，1976）。如Milman等所示，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突变体还携带在回复突变体中被激活的沉默野生型HPRT等位基因。为了测试沉默等位基因是否通过基因组DNA的低甲基化被激活，用DNA甲基化抑制剂处理H23细胞，并通过HAT或阿扎胞苷选择对回复突变体进行评分。在5 µM 5-氮杂胞苷的最佳剂量下，当通过HAT选择测定时，回复频率增加约50倍，当通过氮杂胞苷选择测定时，回复频率增加超过1，000倍。HAT+和阿扎胞苷回复突变体是HPRT杂合子，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，遗传不稳定，回复频率约为10- 4。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，也发现了类似的结果。相比之下，DNA乙基化剂，甲基磺酸乙酯（EMS），并没有增加HAT+回复频率，但是，它确实增加了H23回复突变体的HPRT杂合子恢复为6-硫代鸟嘌呤耐药的频率。在9个EMS回复突变体中，7个缺乏HPRT活性，并且野生型多肽的表达显著降低。这些观察结果支持了DNA甲基化在人类X染色体失活中起重要作用的假设，并且EMS可以通过促进基因组DNA的酶促甲基化来抑制基因表达，如先前在GH 3大鼠垂体瘤细胞中发现的催乳素基因（R. D. Ivarie和J.A. Morris，Proc. Natl. Acad. Sci. USA 79：2967-2970，1982; R. D. Ivarie，J. A. Morris和J. A.马夏尔，摩尔。Cell. 2：179-189，1982）。
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 × 10-8frequency 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 µM 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 10-4frequency. Similar results were found after treatment withN-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).