Cytotoxic and mutagenic responses to X-rays and chemical mutagens in normal and p53-mutated human lymphoblastoid cells

Abstract

To investigate the role of p53 as a guardian of the genome, the mutagenic and cytotoxic responses to mutagens were compared for normal (TK6) and p53-mutated (WTK-1) cells. The characteristics of the mutations that occurred in these cells was also examined. Human lymphoblastoid cell lines TK6 and WTK-1 are derived from the same progenitor cell line, but WTK-1 cells have homozygous p53 mutations resulting in overproduction of mutant p53 protein. The spontaneous mutation frequency at the heterozygous thymidine kinase ( tk) locus in TK6 and WTK-1 cells was 3.5×10 −6 and 101.1×10 −6, respectively. WTK-1 cells were more resistant than TK6 cells to cytotoxic damage by X-rays, ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS), and were more sensitive at the tk locus to the mutagenic effects of X-rays, EMS, MMS and mitomycin C. Molecular analysis of TK mutants by Southern-hybridization demonstrated that 70% of spontaneous mutations and 86% of X-ray induced mutations in TK6 cells resulted from loss of the entire tk allele (loss of heterozygosity; LOH), while 95% of spontaneous and 100% of X-ray induced mutations showed LOH in WTK-1 cells. Densimetric analysis revealed that almost all of the LOH mutants in WTK-1 cells were homozygous at the tk locus, consistent with inter-allelic homologous recombination, or gene conversion. These data indicate that p53-mutated WTK-1 cells are hypermutable, susceptible to some environmental mutagens, and prone to LOH-type gene mutations because of their abnormally high recombinational activity. It may be that genetic instability in p53-mutated cells significantly contribute to the subsequent occurrence of LOH mutations during a multistep tumorigenic process.