Li Gang, Nelsen Caron, Hendrickson Eric A
Department of Molecular Biology, Cellular Biology, and Biochemistry, Brown University, Providence, RI 02912, USA>
Proc Natl Acad Sci U S A. 2002 Jan 22;99(2):832-7. doi: 10.1073/pnas.022649699. Epub 2002 Jan 15.
Ku86 plays a key role in nonhomologous end joining in mammals. Functional inactivation in rodents of either Ku86 or Ku70, which form the heterodimeric DNA end-binding subunit of the DNA-dependent protein kinase complex, is nevertheless compatible with viability. In contrast, no human patient has been described with mutations in either Ku86 or Ku70. This has led to the hypotheses that either these genes are performing an additional essential role(s) and/or redundant pathways exist that mask the phenotypic expression of these genes when they are mutated in humans. To address this issue, we describe here the construction of human somatic cell lines containing a targeted disruption of the Ku86 locus. Human HCT116 colon cancer cells heterozygous for Ku86 were haploinsufficient with an increase in polyploid cells, a reduction in cell proliferation, elevated p53 levels, and a slight hypersensitivity to ionizing radiation. Functional inactivation of the second Ku86 allele resulted in cells with a drastically reduced doubling time. These cells were capable of undergoing only a limited number of cell divisions, after which they underwent apoptosis. These experiments demonstrate that the Ku86 locus is essential in human somatic tissue culture cells.
Ku86在哺乳动物的非同源末端连接中起关键作用。在啮齿动物中,构成DNA依赖性蛋白激酶复合物异二聚体DNA末端结合亚基的Ku86或Ku70功能失活,但仍与生存能力相容。相比之下,尚未描述有人类患者在Ku86或Ku70中发生突变。这导致了这样的假设,即这些基因要么执行额外的重要功能,要么存在冗余途径,在人类中这些基因发生突变时掩盖了它们的表型表达。为了解决这个问题,我们在此描述了构建含有Ku86基因座靶向破坏的人类体细胞系。Ku86杂合的人类HCT116结肠癌细胞单倍体不足,多倍体细胞增加,细胞增殖减少,p53水平升高,对电离辐射略有超敏反应。第二个Ku86等位基因的功能失活导致细胞倍增时间大幅缩短。这些细胞仅能进行有限次数的细胞分裂,之后便会发生凋亡。这些实验表明,Ku86基因座在人类体细胞组织培养细胞中至关重要。