Activation of a nonexpressed hypoxanthine phosphoribosyltransferase allele in mutant H23 HeLa cells by agents that inhibit DNA methylation.

Abstract

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 ,uM 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 Hypoxanthine phosphoribosyltranserferase (HPRT; EC 2.4.2.8) is an enzyme in the salvage pathway of purine biosynthesis in mammals encoded by an X-linked allele (4). The gene has long been used as a model for mutagenesis studies in somatic cell genetics because of the ease by which forward and backward mutations can be selected, its haploid gene dosage, and its involvement in the Lesch-Nyhan syndrome in humans (4). In recent years, it has also been used to implicate DNA methylation in X-chromosome inactivation. In somatic cell hybrids harboring inactive X chromosomes, the HPRT allele has been stably activated by reagents leading to hypomethylation of genomic DNA (13,21,22,26,31 20-fold increased affinity for the mammalian DNA methyltransferase in vitro (9). A major problem of the GH3 cell system, however, was the absence of genetic selection for prolactin mutants. Deficient lines were isolated by random cloning, and phenotypes were scored by biochemical assays. We wanted to know whether the EMS e...

Activation of two new alpha(1,3)fucosyltransferase activities in Chinese hamster ovary cells by 5-azacytidine.

“…Similarly, the mutagens MNNG, ENU, and EMS have different effects on DNA. Although all cause point mutations by chemical modification of DNA (Newbold et al, 1980), ENU and MNNG treatment have been correlated with gene activation and hypomethylation of DNA (Barr et al, 1986;Ivarie and Morris, 1986;MacArthur et al, 1986), whereas EMS can cause gene inactivation and hypermethylation of DNA (Farrance and Ivarie, 1985). Thus, the genetic origins of the different CHO mutants expressing an a(1 ,3)Fuc-T activity (Table 2) may be due to a mutational event, an effect on methylation status, or a DNA rearrangement.…”

Genomic hypomethylation and far-5' sequence alterations are associated with carcinogen-induced activation of the hamster thymidine kinase gene.

“…Some investigations focused on the ability of carcinogens to mutate an active gene and thereby inactivate gene expression or modify the gene product (10,18,26). Recent evidence, however, has expanded the repertoire of carcinogen-induced alterations to include amplification of active genes (34) and activation of quiescent genes (15,23,24).Our laboratory has been investigating carcinogen-induced activation of quiescent genes. To select model systems in which an inactive gene is functionally intact and potentially expressible, we have focused on the class of inactive genes which can be activated by 5-azacytidine (azaCyd), an agent which alters gene expression by epigenetic perturbations presumably related to inhibition of DNA methylation and not by a mutational mechanism (32).…”

“…Some investigations focused on the ability of carcinogens to mutate an active gene and thereby inactivate gene expression or modify the gene product (10,18,26). Recent evidence, however, has expanded the repertoire of carcinogen-induced alterations to include amplification of active genes (34) and activation of quiescent genes (15,23,24).…”