Honma M, Hayashi M, Sofuni T
Division of Genetics and Mutagenesis, National Institute of Health Sciences, Tokyo, Japan.
Mutat Res. 1997 Mar 4;374(1):89-98. doi: 10.1016/s0027-5107(96)00223-0.
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 X 10(-6) and 101.1 X 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.
为了研究p53作为基因组守护者的作用，比较了正常（TK6）细胞和p53突变（WTK - 1）细胞对诱变剂的诱变和细胞毒性反应。还检查了这些细胞中发生的突变特征。人淋巴母细胞系TK6和WTK - 1源自同一祖细胞系，但WTK - 1细胞具有纯合p53突变，导致突变p53蛋白过量产生。TK6和WTK - 1细胞中杂合胸苷激酶（tk）位点的自发突变频率分别为3.5×10⁻⁶和101.1×10⁻⁶。WTK - 1细胞比TK6细胞对X射线、甲基磺酸乙酯（EMS）和甲基磺酸甲酯（MMS）的细胞毒性损伤更具抗性，并且在tk位点对X射线、EMS、MMS和丝裂霉素C的诱变作用更敏感。通过Southern杂交对TK突变体进行分子分析表明，TK6细胞中70%的自发突变和86%的X射线诱导突变是由于整个tk等位基因的缺失（杂合性缺失；LOH），而WTK - 1细胞中95%的自发突变和100%的X射线诱导突变显示出LOH。密度分析显示，WTK - 1细胞中几乎所有的LOH突变体在tk位点都是纯合的，这与等位基因间同源重组或基因转换一致。这些数据表明，p53突变的WTK - 1细胞具有高度的突变性，对某些环境诱变剂敏感，并且由于其异常高的重组活性而易于发生LOH型基因突变。可能是p53突变细胞中的遗传不稳定性在多步骤肿瘤发生过程中显著促进了随后LOH突变的发生。
