Honma M, Momose M, Tanabe H, Sakamoto H, Yu Y, Little J B, Sofuni T, Hayashi M
Division of Genetics and Mutagenesis, National Institute of Health Sciences, Tokyo, Japan.
Mol Carcinog. 2000 Aug;28(4):203-14. doi: 10.1002/1098-2744(200008)28:4<203::aid-mc3>3.0.co;2-1.
Chromosomal double-strand breaks (DSBs) occurring in mammalian cells can initiate genomic instability, and their misrepairs result in chromosomal deletion, amplification, and translocation, common findings in human tumors. The tumor-suppressor protein p53 is involved in maintaining genomic stability. In this study, we demonstrate that the deficiency of wild-type p53 protein may allow unrepaired DSBs to initiate chromosomal instability. The human lymphoblastoid cell line TK6-E6 was established by transfection with human papilloma virus 16 (HPV16) E6 cDNA into parental TK6 cells via a retroviral vector. Abrogation of p53 function by E6 resulted in an increase in the spontaneous mutation frequencies at the heterozygous thymidine kinase (TK) locus but not at the hemizygous hypoxanthine phosphoribosyl transferase (HPRT) locus. Almost all TK-deficient mutants from TK6-E6 cells exhibited loss of heterozygosity (LOH) with the hemizygous TK allele. LOH analysis with microsatellite loci spanning the long arm of chromosome 17, which harbors the TK locus, showed that LOH extended over half of 17q toward the terminal end. Cytogenetic analysis of LOH mutants by chromosome painting indicated a mosaic of chromosomal aberrations involving chromosome 17, in which partial chromosome deletions, amplifications, and multiple translocations appeared heterogeneously in a single mutant. We speculate that spontaneous DSBs trigger the breakage-fusion bridge cycle leading to such multiple chromosome aberrations. In contrast, no chromosomal alterations were observed in TK-deficient mutants from TK6-20C cells expressing wild-type p53. In wild-type p53 cells, spontaneous DSBs appear to be promptly repaired through recombination between homologous chromosomes. These results support a model in which p53 protein contributes to the maintenance of genomic integrity through recombinational repair.
哺乳动物细胞中发生的染色体双链断裂（DSB）可引发基因组不稳定，其错误修复会导致染色体缺失、扩增和易位，这在人类肿瘤中很常见。肿瘤抑制蛋白p53参与维持基因组稳定性。在本研究中，我们证明野生型p53蛋白的缺乏可能使未修复的DSB引发染色体不稳定。人乳头瘤病毒16型（HPV16）E6 cDNA通过逆转录病毒载体转染亲代TK6细胞，从而建立了人淋巴母细胞系TK6-E6。E6对p53功能的废除导致杂合胸苷激酶（TK）位点的自发突变频率增加，但在半合子次黄嘌呤磷酸核糖基转移酶（HPRT）位点则未增加。来自TK6-E6细胞的几乎所有TK缺陷型突变体都表现出半合子TK等位基因的杂合性缺失（LOH）。对跨越17号染色体长臂（该染色体含有TK位点）的微卫星位点进行的LOH分析表明，LOH向末端延伸超过17q的一半。通过染色体涂染对LOH突变体进行细胞遗传学分析表明，存在涉及17号染色体的染色体畸变镶嵌现象，其中部分染色体缺失、扩增和多个易位在单个突变体中呈异质性出现。我们推测自发的DSB触发了断裂-融合桥循环，导致了这种多染色体畸变。相比之下，在表达野生型p53的TK6-20C细胞的TK缺陷型突变体中未观察到染色体改变。在野生型p53细胞中，自发的DSB似乎通过同源染色体之间的重组迅速修复。这些结果支持了一个模型，即p53蛋白通过重组修复有助于维持基因组完整性。
