Azacytidine-induced reactivation of a DNA repair gene in Chinese hamster ovary cells.

Abstract

Six X-ray-sensitive (xrs) strains of the CHO-Kl cell line were shown to revert at a very high frequency after treatment with 5-azacytidine. This suggested that there was a methylated xrs+ gene in these strains which was structurally intact, but not expressed. The xrs strains did not complement one another, and the locus was autosomally located. In view of the frequency of their isolation and their somewhat different phenotypes, we propose that the xrs strains are mutants derived from an active wild-type gene. However, there is in addition a methylated silent gene present in the genome. Azacytidine treatment reactivated this gene. We present a model for the functional hemizygosity of mammalian cell lines, which is based on the inactivation of genes by de novo hypermethylation. In contrast to results with xrs strains, other repair-defective lines were found not to be reverted by azacytidine.A large number of mammalian cell variants were isolated after mutagenic treatments, and there is convincing evidence that many of these are structural gene mutations, comparable to those isolated in various microorganisms (for reviews, see references 2, 7, and 36). Although the cells used were either diploids or originally derived from diploids, recessive autosomal mutants were often isolated at frequencies of 10' or 10-4, suggesting that only a single gene copy is present.This has led to Siminovitch's (36) concept of "functional hemizygosity," in which a substantial part of the genome is effectively haploid. One way this could arise is by chromosome rearrangement and the loss of part of the diploid genome (36).Although there is strong evidence for a genetic basis for stable cell variants, there remains the possibility that heritable variation might be caused by epigenetic alterations in gene activity (15). In support of this theory, it has recently been shown that several enzyme-deficient strains of rodent cells are reverted at very high frequency after treatment with 5-azacytidine (AC) (16)(17)(18)27). This is only a weak mutagen, but is known to be highly effective in reducing DNA methylation, probably through the inhibition of a maintenance methylase (23,40). The results make it likely that these enzyme-deficient strains are epigenetic variants, in which transcription has been shut off by methylation. Considerable independent evidence exists that transcription is associated with hypomethylation, and that certain silent genes, for instance those on the inactive X chromosome or in latent retroviruses, are hypermethylated and can be reactivated by AC (for reviews, see references 8, 11, 22, 32, and 33).We previously described the isolation and partial characterization of six CHO strains which are sensitive to X rays (xrs) and defective in the repair of double strand breaks (21, 25). All of these are recessive traits and fall into one complementation group. We show here that all are reverted to X-ray resistance at very high frequency after AC treatment, indicating that the xrs strains have a copy of the xrs gene inactivated thro...

A DNA end-binding factor involved in double-strand break repair and V(D)J recombination.

“…2C) When xrs5 cells are reverted to X-ray resistance with azacytidine, DEB activity is also restored. Treatment with azacytidine will induce xrs5 cells to revert to X-ray resistance at a frequency of about 1% (20). Azacytidine acts as a competitive inhibitor of methyltransferase to demethylate previously methylated, and therefore silenced, genes.…”

“…Binding activity to the DNA probe f148 (or f320) was measured by a gel electrophoretic mobility shift assay described previously (7,8,17,35 Azacytidine-induced revertants of xrs5. Azacytidine was used to revert xrs5 cells to X-ray resistance as described previously (20). Briefly, 10' cells were seeded into a 25-cm2 flask, allowed to attach, and then grown for 15 h in medium containing 3 ,ug of freshly dissolved 5-azacytidine (Sigma, St. Louis, Mo.)…”

Ku86 Defines the Genetic Defect and Restores X-Ray Resistance and V(D)J Recombination to Complementation Group 5 Hamster Cell Mutants

“…Siminovitch (41) has suggested that a substantial part of the genome in Chinese hamster cell lines is effectively haploid so that many genes are represented by only one functional allele. Functional hemizygosity can arise from the silencing of genes by methylation, and group 5 hamster cell mutants might have two alleles of the XRCC5 gene, with one allele inactivated by mutation and another wild-type allele silenced by methylation (22). RNAs derived from revertants XR-V15B-Rev1 and XR-V15B-Rev2 harbor both wild-type and deleted Ku86 sequences.…”

p53, p16 and cyclin D1: Molecular determinants of radiotherapy treatment response in oral carcinoma

“…25 Similarly, besides being a G1 regulator, p16 has a very important role in cellular senescence. 26 This may be one of the reasons attributed to its independent effect to radiotherapy response.…”