Generation of loss of heterozygosity and its dependency on p53 status in human lymphoblastoid cells

Abstract

Loss of heterozygosity (LOH) is a critical event in the development of human cancers. LOH is thought to result from either a large deletion or recombination between homologous alleles during repair of DNA double-strand breaks (DSBs). These types of genetic alterations produce mutations in the TK gene mutation assay, which detects a wide mutational spectrum, ranging from point mutations to LOH-type mutations. TK6, a human lymphoblastoid cell line, is heterozygous for the thymidine kinase (TK) gene and has a wild-type p53 gene. The related cell lines, TK6-E6 and WTK-1, which are p53-deficient and p53-mutant (Ile237), respectively, are also heterozygous for the TK gene and LOH-type mutation can be detected in these cells. Therefore, comparative studies of TK mutation frequency and spectrum with these cell lines are useful for elucidating the role of p53 in generating LOH and maintaining genomic stability in human cells. We demonstrate here that LOH and its associated genomic instability strongly depend on the p53 status in these cells. TK6-E6 and WTK-1 are defective in the G1/S checkpoint and in apoptosis. Unrepaired DSBs that escape from the checkpoint can potentially initiate genomic instability after DNA replication, resulting in LOH and a variety of chromosome changes. Moreover, genomic instability is enhanced in WTK-1 cells. It is likely that the mutant p53 protein in WTK-1 cells increases LOH in a dominant-negative manner due to its abnormal recombination capacity. We discuss the mutator phenotype and genomic instability associated with p53 inactivation with the goal of elucidating the mechanisms of mutation and DNA repair in untargeted mutagenesis.

Comparison of in vitro micronucleus and gene mutation assay results for p53‐competent versus p53‐deficient human lymphoblastoid cells

“…Because p53 mediates the apoptosis triggered by DNA damage, it seems likely that damaged WTK-1 cells, failing to become apoptotic, survived their damaged state and thereby sustained mutations [Selivanova and Wiman, 1995;Honma, 2005]. In the present comparison of the responses of the same two cell lines, we found some chemicals were cytotoxic at a lower concentration in TK6 cells than in WTK-1 cells and that the difference was most apparent for compounds that induced double strand breaks, such as BLM.…”

“…2). According to the National Institute of Health Sciences laboratory, the historical spontaneous mutant frequency is 2-10 3 10 26 for TK6 cells and 50-250 3 10 26 for WTK-1 cells [Honma, 2005]. In this study, the solvent control mutant frequencies of 93% of the experiments fell within those values.…”

Genotoxicity of Acrylamide and Glycidamide: A Review of the Studies by HPRT Gene and TK Gene Mutation Assays

“…DiŠerent from HPRT gene mutation assay, which detects only intragenic mutations such as point mutations and small deletions, the genetic endpoints detected by the TK mutation assay are multiple, involving point mutations, small deletions, large-scale chromosomal deletions, and so on (50). Moreover, by calculating the relative frequency of two kinds of colonies, or by analyzing the loss of heterozygosity (LOH) in mutants, TK gene mutation test can be used to roughly estimate the cause of genotoxic eŠect.…”

Test No. 490: In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene

“…The first isolated clone, HH4, was mutagenized with ICR191 and a TK heterozygous cell line, TK6, was generated (34). TK6 cells are nearly diploid and the representative karyotype is 47, XY, 13+, t(14; 20), t(3; 21) (35). The human TK locus is located on the long arm of chromosome 17.…”